Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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COMPOSITIONS AND METHODS FOR TREATMENT OF PROTEINOPATHIES
TECHNICAL FIELD
[001] This invention relates to antibody drug conjugates and methods of use
thereof
BACKGROUND OF THE INVENTION
[002] Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's
disease (PD), Huntington's disease (HS), amyotrophic lateral sclerosis (ALS),
prion disease,
inclusion body myositis and various forms of retinal degeneration such as age
related macular
degeneration (AMD) have common cellular and molecular mechanisms including
protein
aggregation, inclusion body formation and oxidative stress leading to
inflammation,
irreversible tissue damage and ultimately death of nerve cells. The aggregates
in these
proteinopathies typically consist of fibers containing misfolded protein with
a beta-sheet
conformation, termed amyloid. Examples of proteins that become misfolded
resulting in
proteinopathies are beta amyloid, tau, alpha synuclein, prion proteins,
superoxide dismutase
(SOD), Huntingtin and serum amyloid A. Amyloid or amyloid-like protein
aggregates are
highly resistant to degradation. P-Amyloid deposits, once formed, are stable
even in the
absence of ongoing amyloid production. Significantly, amyloid or amyloid-like
protein
aggregates catalyze the structural conversion of the normally folded protein
into additional
aggregates via a seeded nucleation-dependent process. Following nucleation,
the ongoing
production of a 'normal' precursor drives additional amyloid formation.
[003] In Alzheimer's disease, AP has a partner in tau protein. Tau
polypeptides
stabilize microtubules. They are abundant in neurons of the central nervous
system and are
less common elsewhere, but are also expressed at very low levels in CNS
astrocytes and
oligodendrocytes. When tau proteins are defective, and no longer stabilize
microtubules
properly, they can cause and/or contribute to proteinopathies (or tauopathies)
such as AD and
frontotemporal dementias. As tau aggregates accumulate, the dendrite is
further sensitized to
A13 induced toxicity - essentially creating a feedback loop whereby tau and
amyloid beta
increasing push one another to become even more active. This leads to greater
aggregation of
tau and amyloid beta and the eventual loss of synaptic function and subsequent
neuronal
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death. In non-AD dementia, mutations in tau protein can also have profound
effects causing
dementia, for example prefrontal dementia.
[004] A novel therapeutic strategy currently under study for AD, AMD and other
proteinopathies is the use of antibody based therapies including active
vaccines and passive
immunotherapy using monoclonal antibodies based on promising preclinical work
in various
disease models including evidence that IgG antibodies can traverse the blood
brain barrier
and bind amyloid plaque. Several mechanisms are postulated to account for the
decrease in
brain amyloid as a result of immunotherapy, including antibody sequestration
of AP in
cerebrospinal fluid and in plaques or in the peripheral circulation,
disaggregation of
oligomers and plaques and other mechanisms that promote the clearance of AP
away from the
brain. The strategy is most developed in AD where several human clinical
trials are being
conducted and data being generated. However, although results from preclinical
work using
amyloid beta immunotherapy were promising, results from human clinical trials
have shown
that while antibodies engage the target and reduce neurodegeneration as
determined by use of
biomarkers, such drugs have failed to produce clinical benefit in patients and
in certain cases
caused brain swelling or inflammation. These data indicate the need for both
earlier
intervention and improved therapies.
[005] Monotherapy may not be effective to treat complex diseases such as AD
and
other proteinopathies. It is likely that the successful treatment of such
diseases will require
the concomitant application of neuroprotective agents. Thus, a number of
factors may limit
the effectiveness of amyloid lowering treatments if applied in isolation.
First, the degree to
which amyloid beta levels need to be reduced to delay onset or slow
progression is unknown.
If amyloid beta concentrations are several-fold above those capable of causing
neuronal
degeneration, a large proportionate reduction in levels might be insufficient
to slow
degeneration. Second, the ideal scenario would include the application of
amyloid beta
lowering immunotherapy drugs in early stages of amyloid beta accumulation,
i.e. years
before onset of symptoms. This approach would require drugs of exceptionally
low toxicity
administered with difficult to achieve high compliance rates years before
clinical
manifestations begin. Third, amyloid-based therapies are unlikely to improve
function or
plasticity of damaged but surviving neurons. Fourth, amyloid associated
proteins such as
apolipoprotein E4 can increase the pathogenicity of the amyloidogenic protein
either by
increasing rate of fibrillogenesis or by other mechanism. Finally, although
the bulk of current
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evidence points to amyloid beta accumulation as a critical primary causative
factor in AD, a
number of other mechanisms might constitute important causative factors as
well. Such non
amyloid beta mechanisms, such as abnormal tau protein, might play synergistic
roles as the
disease progresses. Thus, parallel neuroprotective strategies can play a vital
role in delaying
AD and other proteinopathies and slowing disease progression.
[006] Combination therapy in which different drugs are administered
simultaneously
is a formidable challenge to the pharmaceutical industry from a regulatory
standpoint in
addition to pharmacological and other considerations. Thus, before
investigational new drugs
can be combined into a single therapy, each drug needs to be tested for safety
alone before it
is tested in combination involving a costly clinical trial process. Drugs may
have markedly
different bioavailability and pharmacokinetics such that dosing regimens for
combination
drugs can be cumbersome and even incompatible. Another problem, with
combination
therapies is the need for two different formulations adding to the cost and
complexity of
development ensuring that the formulations are compatible. The interpretation
of data from
clinical trials involving combination therapies with drugs that interact
independently of each
other can be difficult. It would be desirable to overcome these problems
associated with
combination therapies.
[007] Despite advances in understanding mechanisms driving neurodegenerative,
amyloidogenic diseases, such as AD, PD and other proteinopathies, there is a
need for
additional and improved treatments. There is a need, in particular, for
improved therapeutic
reagents and methods for the treatment of proteinopathies, and, in particular,
reagents that can
provide cytoprotective (e.g., neuroprotective) benefits to prevent protein
aggregation, clear
amyloid and counter the detrimental effects of oxidative stress, caused e.g.,
by oxidotoxins,
as well as inhibit Afl and/or tau-induced neuronal toxicity.
SUMMARY OF THE INVENTION
[008] An object of the invention is to provide improved therapeutic reagents
and
methods for the treatment of proteinopathies.
[009] In certain aspects, an object of the invention is to provide antibody-
drug
conjugates (ADC) with neuroprotective properties to reduce inflammation and
oxidative
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stress in the brain, including in cerebrospinal fluid (CSF) and blood
vasculature, in the eye,
and in peripheral tissues such as the kidney and liver.
[0010] An object of the invention is to provide amyloid clearing antibodies
with
neuroprotective properties to reduce inflammation. Another object of the
invention is to
enhance the clearing capacity of an antibody by combining the antibody with a
small
molecule inhibitor of protein aggregation.
[0011] An object of the invention is to provide a high affinity conjugated
antibody
(KDa > 10-9 M) that binds pre-fibrillar amyloid and does not significantly
bind amyloid
precursor proteins.
[0012] An object of the invention is to provide a high affinity conjugated
antibody
(KDa > 10-9M) that binds fibrillar amyloid and does not provoke Fc mediated
phagocytosis or
complement activation.
[0013] An object of the invention is to provide an antibody specific to a
neoepitope in
a target protein, e.g., a free amino or carboxyl group created by cleavage of
a peptide bond in
the precursor protein.
[0014] An object of the invention is to reduce or prevent side effects due to
dissolution of plaques following treatment with immunotherapy or other amyloid
decreasing
drugs, that can lead to vascular deposition of newly released A13 fragments,
which gives rise
to inflammation and vasogenic edema.
[0015] An object of the invention is to provide antioxidant on-site delivery
to
counteract the foci of inflammation leading to vasogenic edema. Another object
of the
invention is to provide cytoprotective agents (e.g., antioxidants) to sites of
inflammation
around pre-existing plaque or resulting from soluble A13 species.
[0016] An object of the invention is to prevent or reduce binding of the
cytoprotective
agent (e.g., antioxidant) to endogenous receptors by conjugating the
cytoprotective agent to
the antibody through the receptor binding site, e.g. for melatonin,
conjugation of N-acety1-5-
methoxytryptamine through Cl of the indole ring thereby preventing binding to
the melatonin
receptor.
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[0017] An object of the invention is to provide compositions comprising an
antibody-
drug conjugate (ADC), said ADC comprising an antibody targeted to an
amyloidogenic
polypeptide or a tau polypeptide conjugated to a cytoprotective agent. In
certain
embodiments, the amyloidogenic polypeptide is an amyloid beta peptide. In
certain
embodiments, the amyloidogenic polypeptide is an amyloid-associated
polypeptide selected
from the group consisting of protease inhibitor alpha 1-antichymotrypsin,
apolipoprotein E
(apoE), and EpoE4. The tau polypeptide may be hyperphosphorylated. In yet
other
embodiments, the amyloidogenic polypeptide is selected from the group
consisting of prion
(PrPSc), amylin, calcitonin, atrial natriuretic factor (AANF), apolipoprotein
Al, serum
amyloid A, medin, transthyretin, lysozyme, beta 2 microglobulin (A132M),
gelsolin,
keratoepithelin, cystatin, immunoglobulin light chain AL, a-synuclein,
Huntingtin, and
superoxide dismutase.
[0018] In certain embodiments, an antibody in an ADC may be humanized. In
certain
embodiments, the antibody is a monoclonal antibody, a humanized antibody, a
chimeric
antibody, a bispecific antibody, an artificial antibody, a scFy antibody or a
F(ab), or fragment
thereof In yet other embodiments, the antibody may be a camel antibody.
[0019] In an embodiment, the cytoprotective agent in an ADC of the invention
is an
antioxidant. An example of an antioxidant is melatonin or analog thereof In
some
embodiments, the antioxidant is selected from the group of antioxidants that
possess one of
more of the following properties: (1) capable of neutralizing free radicals
e.g. hydroxyl
radicals; (2) capable of preventing generation of ROS through metal chelation;
(3) capable of
avoiding the formation of pro-oxidant intermediates; (4) capable of increasing
mitochondrial
metabolism: (5) capable of increasing glucose utilization. Melatonin and
indole-3-propionic
acid are examples of preferable antioxidants based on their multifunctional
antioxidant
activities. Additional antioxidants include but are not limited to the
following group of
compounds and derivatives: an indole amine, an indole acid, vitamin E, vitamin
C, lipoic
acid, uric acid, curcumin, glutathione, a polyphenol, a flavonoid, an
anthraquinone
methylthioninium chloride, dimebone, a rhodamine, an insulin sensitizer e.g.
pioglitazone
and rosiglitazone, an 8-hydroxyquinolone derivative e.g. PBT2, PBT434,
penicillamine,
Trientine, a tetracycline, (N-(pyridin-2-ylmethyl)aniline), and N1,N1-dimethyl-
N4-(pyridin-
2-ylmethyl)benzene-1,4-diamine, 2,6-diaminopyridine. In certain embodiments,
the
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antioxidant is selected from the group consisting of idebenone, cyclohexane-
1,2,3,4,5,6-
hexol, myo-inositol, Scyllo-inositol, and an NO scavenger.
[0020] In certain embodiments the cytoprotective agent is an inhibitor of
protein
aggregation acting through metal binding or intercalating with prefibrillar
protein. Melatonin
is an example of a preferable protein aggregation inhibitor. Thus, melatonin
added to beta
amyloid in the presence of apoE4 results in a potent isoform-specific
inhibitor of fibril
formation, the extent of which is far greater than that of the inhibition
produced by melatonin
alone. Additional antioxidants include but are not limited to the following
group of
compounds and derivatives: AZD-103, cyclohexane-1,2,3,4,5,6-hexol, myo-
inositol, scyllo-
inositol, indole-3-propionic acid, an indole amine, an indole acid, an 8-
hydroxyquinolone
derivative e.g. PBT2, PBT434, penicillamine, Trientine, a tetracycline, (N-
(pyridin-2-
ylmethyl)aniline), N1,N1-dimethyl-N4-(pyridin-2-ylmethyl)benzene-1,4-diamine,
2,6-
diaminopyridine, methylene blue, and TRx0014.
[0021] In certain embodiments the cytoprotective agent lowers production of
amyloid. Useful amyloid lowering compounds include but are not limited to a
gama secretase
inhibitor or modulating factor, a beta secretase inhibitor or modulating
factor, ponatinib.
[0022] In certain embodiments the cytoprotective agent is proneurogenic. Such
proneurogenic compounds include but are not limited to P7C3A20 and P763.
[0023] In certain embodiments, an antibody in an ADC of the invention is
conjugated
to a cytoprotective agent by a linker. Preferably, the linker is selected from
the group
consisting of hydrazone linker, disulfide linker, thioether linker, and
peptide linker. In one
embodiment, the linker is cleavable under intracellular conditions. The
cleavable linker may
be a peptide linker cleavable by an intracellular protease. A peptide linker
can be a dipeptide
linker. The peptide linker can be a citrulline-valine based linker.
[0024] In certain embodiments, the ADC of the invention comprises a marker.
The
marker can be selected from the group consisting of an isotope, a radiolabel,
a fluorescent
label, and an enzyme that catalyzes a detectable modification to a substrate.
The marker can
be conjugated to a cytoprotective agent comprised in the ADC. The marker may
be
incorporated into the cytoprotective agent during synthesis of the
cytoprotective agent.
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[0025] In other embodiments, a pharmaceutical formulation comprising an ADC as
described above, and a pharmaceutically acceptable carrier is provided. In a
specific
embodiment, a pharmaceutical composition comprises an ADC comprising an
antibody
targeted to an amyloidogenic polypeptide or a tau polypeptide conjugated to a
cytoprotective
agent, and a pharmaceutically acceptable carrier.
[0026] In certain embodiments, a method for detecting an amyloid deposit in a
subject is provided, the method comprising administering a composition
comprising an ADC
comprising an antibody targeted to an amyloidogenic polypeptide or a tau
polypeptide
conjugated to a cytoprotective agent and a marker, and detecting the presence
of the marker,
wherein the subject has an amyloid deposit if the marker is detected in the
subject.
[0027] In other embodiments, a method for inhibiting accumulation of an
amyloidogenic polypeptide in the brain of a patient suffering from a
proteinopathy is
provided, the method comprising administering to a patient in need thereof a
therapeutically
effective amount of a composition comprising an ADC, wherein the ADC comprises
an
antibody targeted to an amyloidogenic polypeptide or a tau polypeptide
conjugated to a
cytoprotective agent.
[0028] In another embodiment, a method for promoting clearance of aggregates
from
the brain of a subject is provided, the method comprising administering to the
subject an
ADC-containing composition provided above, such as, e.g., a composition
comprising an
ADC comprising an antibody targeted to a tau polypeptide, under conditions and
in an
amount effective to promote clearance of neurofibrillary tangles from the
brain of the subject.
[0029] In yet another embodiment, a method for treating or delaying onset of a
proteinopathy is provided, the method comprising administering to a subject in
need thereof
an effective amount of an ADC-containing composition provided above, e.g., a
composition
comprising an ADC comprising an antibody targeted to an amyloidogenic
polypeptide or a
tau polypeptide conjugated to a cytoprotective agent, for inhibiting the
formation of fibrils,
the formation of amyloid or amyloid-like deposits, or to inhibit the formation
of
neurofibrillary tangles.
[0030] A proteinopathy for treatment according to any of the methods provided
above
may be selected from the group consisting of age related macular degeneration
(AMD),
glaucoma, traumatic brain injury, cerebral amyloid angiopathy (CAA),
hereditary cerebral
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hemorrhage with amyloidosis Dutch type (HCHWA-D) Alzheimer's disease, early
onset
familial Alzheimer's disease (EOFAD), Down Syndrome, Parkinson's disease (PD),
Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), transmissible
spongiform
encephalopathy, Pick's complex, prion disease, peripheral tissue amyloidosis
(e.g., liver and
kidney), and serum amyloidosis.
[0031] In any of the above embodiments, a subject administered an ADC-
containing
composition of the invention may be a mammal. In a specific embodiment, the
mammal is a
human. In certain embodiments, an ADC-containing composition of the invention,
as
described above is administered intravenously, subcutaneously, intradermally,
intramuscularly, intaperitoneally, intracerebrally, intranasally, orally,
transdermally, buccally,
intra-arterially, intracranially, or intracephalically.
[0032] The details of one or more embodiments of the invention are set forth
in the
description below. Other features, objects, and advantages of the invention
will be apparent
from the description and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] Figure 1 shows antibody-drug conjugates of the invention. In
particular,
Figure lA depicts an antibody conjugated to melatonin using maleimide-based
conjugation
through cysteine side chains on the antibody. Figure 1B depicts an antibody
conjugated to
melatonin via conjugation though lysine side-chains on the antibody via a
Mannich reaction.
Figure 1C depicts an antibody conjugated to a melatonin-PEG derivative using
maleimide-
based conjugation through cysteine side-chains on the antibody. Figure 1D
depicts an
antibody conjugated to melatonin via maleimide-based conjugation through
lysine side chains
on the antibody modified with 2-iminothiolane.
[0034] Figure 2 shows the results of experiments testing the capacity of
constructs of
the invention to reduce P-amyloid fibrillogenisis as evaluated by Thioflavin T
Spectrofluorometry. In particular, Figure 2A shows florescence intensities of
tested samples
measured at an excitation wavelength of 450 nm and an emission wavelength of
485 nm at
t=0. Figure 2B shows florescence data at t=0 normalized as a percentage of a
control sample.
Figure 2C shows florescence intensities of tested samples measured at an
excitation
wavelength of 450 nm and an emission wavelength of 485 nm at t=24 hours.
Figure 2D
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shows a time scan of fluorescence intensity at t=24 hours normalized as a
percentage of a
control sample.
DETAILED DESCRIPTION
I. Overview
[0035] Improved compositions and methods are needed for the treatment of
neurodegenerative diseases such as, but not limited to, Alzheimer's disease
(AD), Parkinson's
disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS)
and prion
diseases, diseases which are referred to herein generally as proteinopathies.
Certain types of
neuroprotective molecules that have antioxidant or anti-aggregation properties
can block beta
amyloid neurotoxicity by a variety of mechanisms including metal-chelation,
free radical
scavenging activity, mitochondrial activation, insulin sensitization and other
mechanisms. For
example the antioxidant melatonin can interact with the high affinity copper
binding site in
AP and inhibit fibril formation, and can provide cytoprotective effects to
neuronal cells in AD
and other proteinopathies. See, U.S. Pat. No. 5,958,964.
[0036] The
invention is based in part on the discovery that antibody-drug
conjugates (termed "ADC" herein) of an antioxidant molecule (or molecule that
inhibits
aggregation) and antibody directed to an antibody targeted to a specific
polypeptide involved
in the etiology of a proteinopathy are improved therapeutics because the
amyloid clearing
antibody is thereby equipped with additional neuroprotective properties to
reduce oxidative
damage and in some cases also inhibit aggregation or promote disaggregation.
As one
example, and without limitation, melatonin, when conjugated to an antibody
targeted to a
specific polypeptide involved in the etiology of a proteinopathy (e.g.,
amyloid P peptide or
Tau), can provide an effective therapeutic reagent. Without being bound by
theory, ADC can
recognize and remove toxic amyloidogenic polypeptides and polypeptides that
cause
neurofibrillary tangles (e.g., tau) while also providing cytoprotective
benefits to the target
cells (e.g., dendrites or other neuronal cells). Thus, in certain embodiments,
a composition
comprising an antibody which is targeted to an amyloid polypeptide (e.g.,
amyloid 13 peptide)
or to tau or another misfolded protein, wherein said antibody is conjugated to
a cytoprotective
agent (e.g., an antioxidant such as melatonin), is provided.
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[0037] In a specific embodiment, an ADC of the invention comprises an A13
targeted
antibody conjugated to melatonin. In another embodiment, an ADC of the
invention
comprises a tau polypeptide-targeted antibody conjugated to melatonin. In some
instances
antibody in the ADC is preferably conjugated to melatonin via a linker, such
as, but not
limited to a hydrazone linker, disulfide linker, thioether linker, or peptide
linker.
Alternatively, antibody in the ADC is preferably conjugated to melatonin via
solvent exposed
lysine residues using maleimide based linkers (see experimental data) or using
a Mannich
type reaction.
[0038] Also provided are methods for inhibiting accumulation of amyloid 13
peptide
or hyperphosphorylated or cleaved tau polypeptide in the brain of a patient
suffering from a
proteinopathy, comprising contacting amyloid 13 peptide or hyperphosphorylated
tau
polypeptide in the brain of the patient with an ADC composition provided
herein.
[0039] In yet another embodiment, a method for treating or delaying onset of a
proteinopathy (e.g., age related macular degeneration (AMD), glaucoma,
traumatic brain
injury, cerebral amyloid angiopathy (CAA), hereditary cerebral hemorrhage with
amyloidosis
Dutch type (HCHWA-D) AD, early onset familial AD (EOFAD), Down Syndrome,
Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral
sclerosis (ALS),
prion disease, etc.), comprising administering to a subject in need thereof an
effective amount
of an ADC composition according to the present invention, to inhibit the
formation of fibrils
or the formation of amyloid or amyloid-like deposits associated with
amyloidosis-related
diseases, or to inhibit the formation of neurofibrillary tangles associated
with tauopathies.
Non-neuropathic amyloidogenic diseases and disorders may also be treated
according to the
methods provided herein, as described in detail, infra.
[0040] Alzheimer's disease (AD) is a dementing illness with progressive loss
of
memory, task performance, speech and recognition of people and objects. There
is extensive
degeneration of neurons especially in the basal forebrain and hippocampus. At
least as
important for pathogenesis may be the synaptic pathology and altered neuronal
connections.
Pathological manifestations of AD include extracellular plaques of 13-amyloid
and
intracellular neurofibrillary tangles composed of abnormally bundled
cytoskeletal fibers. AD
involves two major kinds of protein aggregates. Extracellular aggregates known
as neuritic
plaques are comprised primarily of the beta amyloid peptide, which is derived
by the
proteolytic processing of the amyloid precursor protein, APP. The beta amyloid
peptides have
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beta sheet structures. There are also intracellular aggregates of the
microtubule associated
protein ¨ tau. The deposition of amyloid plaques is thought to destabilize
neurons by
mechanisms which require further clarification. Tangles are associated with
hyperphosphorylation of tau, a microtubule-associated protein, and of
neurofilament H/M
subunits, processes that lead to misfolding and accumulation of these
proteins, along with a
disruption of microtubules.
[0041] Deposition of cerebral amyloid is a primary neuropathologic marker of
AD.
Thisamyloid is composed of a 40-42 amino acid peptide called the amyloid beta
protein (AP).
Amyloid deposits in AD are found mainly as components of senile plaques, and
in the walls
of cerebral and meningeal blood vessels, where they gradually become
dystrophic and cause
damage through inflammation and oxidative stress. In addition, various soluble
toxic AP
species, including oligomers and fibrils, are found in cerebrospinal fluid. As
the
concentration of fibrils increases, they eventually deposit as plaque on the
surface of cells.
[0042] The discovery that AD could be inherited in an autosomal dominant
fashion
due to a mutation in the gene encoding APP was a seminal event in AD research.
Amyloid-P
peptide is excised from APP via sequential scission by the 3-APP cleaving
enzyme (BACE)
and 7- secretase. These observations led to the articulation of the amyloid
cascade hypothesis.
This hypothesis was further supported by the discovery that AD could also be
caused by
autosomal dominant mutations in presenilin 1 (PSEN1) and PSEN2, which are both
homologous proteins that can form the catalytic active site of gamma-
secretase.
[0043] In Alzheimer's disease, A13 has a partner in tau protein. Tau
polypeptides
stabilize microtubules. They are abundant in neurons of the central nervous
system and are
less common elsewhere, but are also expressed at very low levels in CNS
astrocytes and
oligodendrocytes. When tau proteins are defective, and no longer stabilize
microtubules
properly, they can cause and/or contribute to proteinopathies (or tauopathies)
such as AD and
frontotemporal dementias. In the early phase, neurofibrillary tangles
accumulate around the
dendrite and begin to damage the dendrite. Although there is much evidence
linking tau to
neurodegeneration, the precise mechanism of tau-mediated neurotoxicity remains
to be
elucidated. For many years, it was assumed that NFTs were the cause of
neuronal toxicity.
However, in some animal models overexpressing tau, neurodegeneration has been
demonstrated in the absence of overt NFT pathology. Additionally, recent
evidence suggests
that memory function and neuronal loss can be restored in a tauopathy mouse
model despite
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the ongoing accumulation of NFTs. Moreover, NFTs have been suggested to
persist in
neurons for 20-30 years making them unlikely candidates for catalysing
immediate toxicity.
In fact, a large immunohistochemical study on cholinergic basal forebrain
neurons in the
nucleus basal is using an early tau marker demonstrated pre-tangle neurons,
and NT staining
correlates extremely well with cognitive decline, which occurs before the
emergence of
significant NFT pathology. Finally, synaptic loss correlates better with
cognitive decline than
NFTs, again suggesting the possibility of different mechanisms for tau
toxicity: At least two
forms of pathologic tau preceded tangle formation, oligomers which occur at
the very earliest
stages of the disease and a truncated form "delta tau" which is produced by
caspase cleavage
on the intact protein.
[0044] As tau aggregates accumulate, the dendrite is further sensitized to A13
induced
toxicity - essentially creating a feedback loop whereby tau and amyloid beta
increasing push
one another to become even more active. This leads to greater aggregation of
tau and amyloid
beta and the eventual loss of synaptic function and subsequent neuronal death.
In non-AD
dementia, mutations in tau protein can also have profound effects causing
dementia, for
example prefrontal dementia.
[0045] A number of neurological diseases are known to have filamentous
cellular
inclusions containing microtubule associated protein tau, e.g., Alzheimer's
disease (AD),
progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's
disease (PiD)
and a group of related disorders collectively termed frontotemporal dementia
with
Parkinsonism linked to chromosome 17 (FTDP-17), amyotropic lateral sclerosis
(ALS),
Creutzfeldt-Jakob disease (CJD), dementia pugilistica (DP),
GerstmannStraussler- Scheinker
disease (GSSD), Lewy body disease and Huntington disease. Although the
etiology, clinical
symptoms, pathologic findings and the biochemical composition of inclusions in
these
diseases are different, there is emerging evidence suggesting that the
mechanisms involved in
aggregation of normal cellular proteins to form various filamentous inclusions
are
comparable. It is believed, that an initial alteration in conformation of
microtubule associated
protein tau, which initiates generation of nuclei or seeds for filament
assembly, is one of the
key features. This process can be influenced by the posttranslational
modification of normal
proteins, by mutation or deletion of certain genes and by factors that bind
normal proteins and
thus alter their conformation.
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[0046] Inheritance of apoE4 is a strong risk factor for the development of
late-onset
sporadic Alzheimer's disease. Several lines of evidence suggest that apoE4
binds to the
Alzheimer Abeta protein and, under certain experimental conditions, promotes
formation of
beta-sheet structures and amyloid fibrils.
[0047] AD pathology has many similarities to changes that are responsible for
retinal
degeneration including glaucoma, diabetic retinopathy, Bests disease and both
dry and wet
forms of age related macular degeneration. An important characteristic common
to between
AD and AMD for example, is the presence of amyloid 13 in the senile plaques of
the AD brain
and in the drusen of AMD patients. A13 is a key regulator of the progression
from drusen to
AMD causing an imbalance of angiogenesis-related factors in the retinal
pigment epithelial
(RPE) cells. Mice that lack the A13-degrading enzyme neprilysin develop RPE
degeneration,
and the sub-RPE deposits that are formed have features similar to those of AMD
in humans.
Moreover, changes in the concentrations of A13 and Tau in the vitreal fluid of
the eye mirror
changes in the cerebral spinal fluid (CSF) of Alzheimer's patients where A13
reduction is
accompanied by a concomitant increase in tau protein. These data suggest that
a common
pathogenic mechanism exist between AMD and AD. Thus, therapeutic approaches
that have
targeted A13 in patients with AD can also be applied to AMD and possibly other
forms of
retinal degeneration.
[0048] Parkinson's disease (PD) is characterized by resting tremor, rigidity,
slow
movements and other features such as postural and autonomic instability. It is
caused by
degeneration of dopaminergic neurons in the substantia nigra of the midbrain
and other
monoaminergic neurons in the brain stem. The discovery of several genes in
which mutations
cause early onset forms of PD has greatly accelerated research and led to a
better
understanding of the disease. Point mutations or increased gene dosage of the
alpha synuclein
gene cause autosomal dominant PD by a gain of function mechanism. Recessive
early onset
PD can be caused by point mutations in the genes encoding parkin, DJ1 or
PINK1,
presumably by loss of function mechanisms. The pathological hallmark feature
of adult PD is
the Lewy Body, an inclusion found in cytoplasm of neurons. Lewy Bodies are
densest in the
substantia nigra but can also be present in monoaminergic, cerebral cortical
and other
neurons. There are also aggregates in neuritis, which are referred to as Lewy
neurites. A
major constituent of Lewy bodies is aggregated alpha synuclein protein.
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[0049] Huntington disease (HD) is a progressive neurodegenerative disorder
caused
by expansion of CAG repeat coding for polyglutamine in the N-terminus of the
huntingtin
protein. There is a striking threshold between the threshold for aggregation
in vitro and
threshold for disease in humans. Inclusions containing Huntingtin are present
in regions of
the brain that are most degenerate. Huntingtin aggregates can be labeled with
antibodies to
the N-terminus of the protein, or antibodies to ubiquitin, a marker of
misfolded proteins and a
signal for degradation by the proteasome. Proteasomes may have difficulty
digesting them,
however, leading to the accumulation of aggregates. The aggregates contain
fibers and appear
to have a beta sheet characteristic of beta amyloid. Other proteins such as
Creb binding
protein may be recruited into Huntingtin aggregates.
[0050] Amyotrophic lateral sclerosis (ALS) is a progressive fatal disease
caused by
degeneration of lower motor neurons in the horn of the spinal cord and upper
motor neurons
of the cerebral cortex, resulting in progressive motor weakness. Rare early
onset familial
forms of the disorder can be caused by mutations in the superoxide dismutase
gene which
cause aggregation of the protein in inclusions.
[0051] Neurodegenerative diseases caused by prions can be sporadic or can be
acquired by either environmental transmission or via genetic mutations.
Pathology can
include amyloid plaques which appear similar to those of AD except that they
are formed by
prion proteins. Prion aggregation can take place both extracellularly and
intracellularly.
[0052] The important role of oxidative stress, an imbalance between the
production
and detoxification of oxidative reaction products continues to be the subject
of extensive
research in AD. While oxidation products accumulate to some degree in normal
brain, their
levels increase with age and are substantially higher in AD, including in its
early stages.
Excessive levels of hydrogen peroxide and ROS such as hydroxyl free radical
and superoxide
lead to the formation of oxidation products including oxidized proteins, lipid
peroxides,
advanced glycosylation end products and DNA adducts. Protein and lipid
oxidation leads to
loss of critical enzyme functions, including those regulating glutamate
transport, which
results in excitotoxicity due to excessive extracellular glutamate, and to the
loss of ATPases,
causing disruption of calcium homeostasis and impaired mitochondrial function.
Oxidative
stress triggers degenerative signaling, including activation of stress kinases
and caspases.
Sources of oxidative stress in AD include impaired mitochondrial metabolism,
decreases
glucose utilization and A13. The peptide may be a major source of ROS when
binding Cu2+
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or Fe3+ and also by interacting with RAGE receptor at the cell surface to
promote lipid
peroxidation.
[0053] With regard to oxidative stress, the pro-oxidant properties of the free
amyloid-
13 molecule (AP) may lead to hydroxyl radical-induced cell death (e.g., by
Fenton and Fenton-
like reactions). Additionally, AP initiates flavoenzyme-dependent rises in
intracellular H202
and lipid peroxides, which also cause radical generation. Rises in AP protein
have been
shown to induce oxidative stress. Impairment of neurotrophin activity on
associated tyrosine
kinase receptors has been suggested to represent an important factor in AD
pathology.
Moreover, the pathophysiological phenomena associated with AD have also been
found to be
associated with other neurodegenerative disorders such as Parkinson's disease
(PD),
Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), prion
diseases, Pick's
complex, and other proteinopathies. See, Srinivasan et al. (2006) Behavioral
and Brain
Functions, 2:15; Ross and Poirier (2004) Nat Med Rev; 510-517.
II. Definitions
[0054] As used herein, the term "amyloidogenic polypeptide" means a
polypeptide
that is capable of forming amyloid fibrils, filaments, and/or amyloid
deposits, such as, but not
limited to, amyloid 13 peptide (AP), amylin, calcitonin, atrial natriuretic
factor, apolipoprotein
AT, serum amyloid A, etc. Amyloid fibrils are insoluble proteinaceous
fibrillar aggregates
with a characteristic structure (the cross-3 core) that form and deposit in
more than 40
pathological conditions in humans. As used herein, "amyloid beta peptide" (AP)
refers to the
40-42 amino acid peptide that makes up the cerebral amyloid which is the
primary
neuropathologic marker of AD and other amyloidogenic diseases, and includes
fragments of
AP capable of causing cytotoxic effects on cells. For example, one such
fragment of AP is the
fragment made of up amino acid residues 25-35 of AP. See, Glenner, G. G., and
Wong, C. W.
(1984) Biochem Biophys Res Commun 120:885-890, for the full amino acid
sequence of AP.
[0055] As used herein, "amyloid-associated polypeptide" includes polypeptides
that
may be found in amyloid deposits, such as, but not limited to, the protease
inhibitor alpha 1-
antichymotrypsin and the lipid transport protein apolipoprotein E (apoE),
which are
intimately associated with AP, e.g., in the filamentous amyloid deposits of
AD, and also
includes amyloid-promoting factors (pathological chaperones) such as, but not
limited to,
EpoE4.
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[0056] As used herein, the term "antibody" means an immunoglobulin protein,
which
is capable of binding an antigen, and includes full length antibodies,
monoclonal antibodies
(including full length monoclonal antibodies), polyclonal antibodies, multi-
specific
antibodies (e.g., bispecific antibodies), human, humanized or chimeric
antibodies, unibody
and antibody fragments, e.g., Fab fragments, F(ab'), Fab', F(ab')2, and Fy
fragment,
fragments produced by a Fab expression library, epitope-binding fragments of
any of the
above, and engineered forms of antibodies, e.g., scFy molecules, so long as
they exhibit the
desired activity, e.g., binding to the desired target. The term also includes
single chain
antibodies in which heavy and light chain variable domains are linked through
a spacer. Thus,
for example, non-limiting examples of an antibody according to the present
invention include
an antibody which is targeted to (i.e., capable of binding to) an amyloid
polypeptide (e.g.,
amyloid p peptide), to an amyloid-associated polypeptide, or to tau. An
antibody that is
"targeted to" a polypeptide, as used herein, is an antibody that specifically
recognizes and
binds to that polypeptide.
[0057] The term "humanized antibody" means an antibody in which the
complementary-determining regions (CDRs) of a mouse or other non-human
antibody are
grafted onto a human antibody framework. By human antibody framework is meant
the entire
human antibody excluding the CDRs.
[0058] The term "chimeric antibody" refers to an antibody in which the whole
of the
variable regions of a mouse or rat antibody are expressed along with human
constant regions.
[0059] The term "free end specific" means a molecule which preferentially
binds to a
particular free terminus/end of a peptide or protein. For example, in the
context of an
amyloid p peptide, the term "free end specific" refers to binding specifically
to a free
terminus of an amyloid 13 peptide or to any fragment thereof to slow down or
prevent the
accumulation of amyloid 13 peptides in the extracellular space, interstitial
fluid and/or
cerebrospinal fluid and to block the interaction of A13 peptides with other
molecules that
contribute to the neurotoxicity of A13. The term "free end specific" can be
used in reference
to any amyloidogenic protein fragment, including, for example, the C-terminal
portion of
delta Tau, Huntingtin cleavage products, Abeta protein, and the like.
[0060] As used herein, the term "cytoprotective agent" refers to an agent that
is
capable of ameliorating one or more deleterious effects in a cell, such as,
e.g., a neuronal cell,
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that are caused by oxidotoxins, oxidative stress (e.g., caused by amyloid 13
and/or
mitochondrial dysfunction), and/or one or more other processes associated with
proteinopathies (i.e., diseases or disorders associated with protein
misfolding and/or amyloid
deposition and/or neurofibrillary tangles). By way of example, and without
limitation, a
cytoprotective agent can be an antioxidant, such as, but not limited to,
melatonin, an indole
acid, an indole amine, an indole acid, vitamin E, vitamin C, lipoic acid, uric
acid, curcumin,
glutathione, a polyphenol, a flavonoid, an anthraquinone methylthioninium
chloride,
dimebone, a rhodamine, an insulin sensitizer e.g. pioglitazone and
rosiglitazone, an 8-
hydroxyquinolone derivative e.g. PBT2, PBT434, penicillamine, Trientine, a
tetracycline, (N-
(pyridin-2-ylmethyl)aniline), N1,N1-dimethyl-N4-(pyridin-2-ylmethyl)benzene-
1,4-diamine,
2,6-diaminopyridine, idebenone, cyclohexane-1,2,3,4,5,6-hexol, myo-inositol,
Scyllo-
inositol, NO scavengers, and other antifibrillogenic molecules.
[0061] As further used herein, "melatonin" refers to the compound N-[2-(5-
Methoxyindo1-3-yl)ethyl]acetamide (also referred to as N-acetyl-5-
methoxytryptamine).
Melatonin analogs that retain the function of preventing the cytotoxic effects
of AP are well
known in the art. Melatonin and such analogs are described in greater detail,
infra.
[0062] As used herein, the term "hydrazine linker" refers to a linker moiety
that, upon
a change in condition, such as a shift in pH, will undergo a cyclization
reaction and form one
or more rings. The hydrazine moiety is converted to a hydrazone when attached.
This
attachment can occur, for example, through a reaction with a ketone group on
the L moiety.
Therefore, the term "hydrazone linker" can also be used to describe the
hydrazine linkers of
the invention.
[0063] Within the meaning of the present invention, the term "inhibit" and its
grammatical variations are used to refer to any level of reduction in a
function or amount. For
example, when used in relation to "inhibiting accumulation of amyloid P
polypeptides or
amyloid-associated polypeptides" in a subject, "inhibit" means any level of
reduction of the
number of any species of amyloid P polypeptides in the subject and/or any
level of reduction
of the size and/or frequency of amyloid deposits in the subject.
[0064] As used herein, the terms "treat", "treatment", and the like mean to
relieve or
alleviate at least one symptom associated with a condition (e.g.,
proteinopathy), or to slow or
reverse the progression of such condition. For example, in relation to AP
related diseases
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such as, but not limited to, AD, PD, or HD, the term "treat" may mean to
relieve or alleviate
at least one symptom of the disease, such as, for example, and without
limitation, memory
loss and/or confusion, inability to recognize family and friends, inability to
learn new things,
difficulty carrying out tasks that involve multiple steps (such as getting
dressed), problems
coping with new situations, delusions and paranoia, and/or impulsive behavior
(for AD);
involuntary trembling, rigid or stiff muscles, loss of ability to make rapid,
spontaneous
movements, gait characterized by bent or flexed body, difficult in maintaining
balance (for
PD); movement disorders such as involuntary jerking or writhing movements
(chorea),
involuntary, sustained contracture of muscles (dystonia), muscle rigidity,
slow, uncoordinated
fine movements, slow or abnormal eye movements, impaired gait, posture and
balance,
difficulty with the physical production of speech, and/or difficulty
swallowing and cognitive
disorders, such as difficulty planning, organizing and prioritizing tasks,
inability to start a
task or conversation, lack of flexibility, or the tendency to get stuck on a
thought, behavior or
action (perseveration), lack of impulse control that can result in outbursts,
acting without
thinking and sexual promiscuity, problems with spatial perception that can
result in falls,
clumsiness or accidents, lack of awareness of one's own behaviors and
abilities, difficulty
focusing on a task for long periods, slowness in processing thoughts or
"finding" words,
and/or difficulty in learning new information (for HD). Those of skill in the
art will
appreciate that the symptoms of the proteinopathies that can be treated
according to the
present invention are well known in art, and the foregoing are provided by way
of non-
limiting example. The skilled artisan (e.g., an individual's physician) will
understand whether
one or more symptoms of a proteinopathy contemplated for treatment herein have
been
treated upon examination of the individual.
[0065] In certain aspects, the compounds and compositions described herein may
be
used to delay the onset and/or reduce the risk of developing or worsening a
disease. For
delayed onset of a proteinopathy, as provided herein, preferably, an ADC of
the invention
will delay the onset of the disease (i.e., appearance of clinical
manifestation of the disease, or
appearance of increased symptoms of the disease after initial (early) signs of
the disease are
observed, e.g., by the subject or the subject's physician) by at least 1 year,
at least 2 years, at
least 3 years, at least 4 years, at least 5 years, at least 6 years, at least
7 years, or longer. Thus,
for example, if a subject is suspected to be in early stages of AD or at risk
of developing AD
at the age of 61, treatment of the subject with an ADC or ADC-containing
composition of the
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invention, preferably, although not necessarily, would delay the onset at
least until the age of
62 or older.
[0066] As used herein, the term "therapeutically effective" applied to dose or
amount
refers to that quantity of a compound or composition (e.g., pharmaceutical
composition) that
is sufficient to result in a desired activity upon administration to an animal
in need thereof
Thus, within the context of the present invention, the term "therapeutically
effective amount"
refers to that quantity of a compound or composition that is sufficient to
treat at least one
symptom of a proteinopathy, as described above. When a combination of active
ingredients is
administered, a therapeutically effective amount of the combination may or may
not include
amounts of each ingredient that would have been effective if administered
individually. A
therapeutically effective amount does not necessarily occur by administration
of one dose,
and may occur only after administration of a series of doses. Thus, a
therapeutically effective
amount may be administered in one or more administrations.
[0067] As used herein "combination therapy" or "adjunct therapy" means that
the
individual in need of the ADC-containing composition is treated or given
another drug or
therapeutic agent ("21d composition") for the disease in conjunction with the
ADC-containing
composition. Combination therapy can be sequential therapy where the
individual (e.g.,
patient) is treated first with one composition and then the second
composition, or the two are
given simultaneously. These compositions are said to be "coadministered."
[0068] The phrase "pharmaceutically acceptable" refers to molecular entities
and
compositions that are physiologically tolerable when administered to a human.
Preferably, as
used herein, the term "pharmaceutically acceptable" means approved by a
regulatory agency
of the Federal or a state government or listed in the U.S. Pharmacopeia or
other generally
recognized pharmacopeia for use in animals, and more particularly in humans.
[0069] The terms "express" and "expression" mean allowing or causing the
information in a gene or DNA sequence to become manifest, for example,
producing an non-
coding (untranslated) RNA or a protein by activating the cellular functions
involved in
transcription and translation of a corresponding gene or DNA sequence. A DNA
sequence is
expressed in or by a cell to form an "expression product" such as RNA or a
protein. The
expression product itself, e.g. the resulting RNA or protein, may also be said
to be
"expressed" by the cell.
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[0070] The terms "percent (%) sequence similarity", "percent (%) sequence
identity",
and the like, generally refer to the degree of identity or correspondence
between different
nucleotide sequences of nucleic acid molecules or amino acid sequences of
proteins that may
or may not share a common evolutionary origin (see Reeck et al., supra).
Sequence identity
can be determined using any of a number of publicly available sequence
comparison
algorithms, such as BLAST, FASTA, DNA Strider, GCG (Genetics Computer Group,
Program Manual for the GCG Package, Version 7, Madison, Wisconsin), etc.
[0071] To determine the percent identity between two amino acid sequences or
two
nucleic acid molecules, the sequences are aligned for optimal comparison
purposes. The
percent identity between the two sequences is a function of the number of
identical positions
shared by the sequences. In one embodiment, the two sequences are, or are
about, of the same
length. The percent identity between two sequences can be determined using
techniques
similar to those described below, with or without allowing gaps. In
calculating percent
sequence identity, typically exact matches are counted.
[0072] The determination of percent identity between two sequences can be
accomplished using a mathematical algorithm. A non-limiting example of a
mathematical
algorithm utilized for the comparison of two sequences is the algorithm of
Karlin and
Altschul, Proc. Natl. Acad. Sci. USA 1990; 87:2264, modified as in Karlin and
Altschul,
Proc. Natl. Acad. Sci. USA 1993; 90:5873-5877. Such an algorithm is
incorporated into the
NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 1990; 215:403.
BLAST
nucleotide searches can be performed with the NBLAST program, score = 100,
word length
= 12, to obtain nucleotide sequences homologous to sequences of the invention.
BLAST
protein searches can be performed with the XBLAST program, score = 50, word
length = 3,
to obtain amino acid sequences homologous to protein sequences of the
invention. To obtain
gapped alignments for comparison purposes, Gapped BLAST can be utilized as
described in
Altschul et al., Nucleic Acids Res. 1997;25:3389. Alternatively, PSI-Blast can
be used to
perform an iterated search that detects distant relationship between
molecules. See Altschul et
al. (1997), supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs,
the
default parameters of the respective programs (e.g., XBLAST and NBLAST) can be
used.
See ncbi.nlm.nih.gov/BLAST/ on the WorldWideWeb. Another non-limiting example
of a
mathematical algorithm utilized for the comparison of sequences is the
algorithm of Myers
and Miller, CABIOS 1988; 4:11-17. Such an algorithm is incorporated into the
ALIGN
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program (version 2.0), which is part of the GCG sequence alignment software
package. When
utilizing the ALIGN program for comparing amino acid sequences, a PAM120
weight
residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
[0073] In accordance with the present invention there may be employed
conventional
molecular biology, microbiology, and recombinant DNA techniques within the
skill of the
art. Such techniques are explained fully in the literature. See, e.g.,
Sambrook, Fritsch &
Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring
Harbor,
NY: Cold Spring Harbor Laboratory Press, 1989 (herein "Sambrook et al.,
1989"); DNA
Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985);
Oligonucleotide
Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization [B.D. Hames & S.J.
Higgins eds.
(1985)]; Transcription And Translation [B.D. Hames & S.J. Higgins, eds.
(1984)]; Animal
Cell Culture [R.I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL
Press,
(1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); Ausubel,
F.M. et al.
(eds.). Current Protocols in Molecular Biology. John Wiley & Sons, Inc., 1994.
These
techniques include site directed mutagenesis as described in Kunkel, Proc.
Natl. Acad. Sci.
USA 82: 488- 492 (1985), U. S. Patent No. 5,071, 743, Fukuoka et al. ,
Biochem. Biophys.
Res. Commun. 263: 357-360 (1999); Kim and Maas, BioTech. 28: 196-198 (2000);
Parikh
and Guengerich, BioTech. 24: 4 28-431 (1998); Ray and Nickoloff, BioTech. 13:
342-346
(1992); Wang et al., BioTech. 19: 556-559 (1995); Wang and Malcolm, BioTech.
26: 680-
682 (1999); Xu and Gong, BioTech. 26: 639-641 (1999), U.S. Patents Nos. 5,789,
166 and
5,932, 419, Hogrefe, Strategies 14. 3: 74-75 (2001), U. S. Patents Nos.
5,702,931, 5,780,270,
and 6,242,222, Angag and Schutz, Biotech. 30: 486-488 (2001), Wang and
Wilkinson,
Biotech. 29: 976-978 (2000), Kang et al., Biotech. 20: 44-46 (1996), Ogel and
McPherson,
Protein Engineer. 5: 467-468 (1992), Kirsch and Joly, Nuc. Acids. Res. 26:
1848-1850
(1998), Rhem and Hancock, J. Bacteriol. 178: 3346-3349 (1996), Boles and
Miogsa, Curr.
Genet. 28: 197-198 (1995), Barrenttino et al., Nuc. Acids. Res. 22: 541-542
(1993), Tessier
and Thomas, Meths. Molec. Biol. 57: 229-237, and Pons et al., Meth. Molec.
Biol. 67: 209-
218.
III. Antibody Drug Conjugates (ADC)
(A) Antibodies
[0074] Antibodies encompassed by the present invention may target any
polypeptides
involved in a proteinopathy. For example, amyloidogenic polypeptides involved
in plaque
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deposits and fibril formation (e.g., A13), amyloid associated proteins (e.g.
al-
antichymotrypsin, Apolipoprotein E, apoE, and Creb Binding Proteins), tau, as
well as prions,
are suitable targets for antibodies according to the present invention. Other,
non-limiting
examples of antibody targets encompassed by the present invention, include for
example,
TAPP (Amylin), calcitonin, atrial natriuretic factor (AANF), apolipoprotein AT
(AApoA1),
serum amyloid A (AA), medin (AMed), transthyretin (ATTR), lysozyme (ALys),
beta 2
microglobulin (A132M), Gelsolin (AGel), keratoepithelin (AKer), beta amyloid
(A13), cystatin
(ACys), immunoglobulin light chain AL (AL), tau polypeptides, a-synuclein,
Huntingtin, and
superoxide dismutase. Further, suitable targets include whole protein and
protein fragments
and post-translational modifications, e.g., phosphorylated sites. See,
Southwell and Patterson
(2010) Reviews in the Neurosciences 21, 273-287.
[0075] An antibody according to the present invention may be targeted, for
example,
to the amino terminus or the carboxy terminus of the target polypeptide, or
the antibody may
be targeted to a mid domain of the target polypeptide. The antibody may also
be a
conformational antibody (i.e., may recognize a specific structure of the
folded amyloid or
amyloid associated polypeptide). The antibody may recognize any part or
conformation of a
target polypeptide. Non-limiting examples of antibodies specific for (targeted
to) amyloid
beta include, for example, bapineuzumab (see,
http ://www. alzforum. org/drg/drc/detail. asp? id=101), P
onezumab (see,
http ://www. alzforum. org/drg/drc/detail. asp? id=139),
gantenerumab (see,
http ://www. alzforum. org/new/detail . asp? id=2933 ), s
olaneszumab, (see,
http ://www. alzforum. org/drg/drc/detail. asp? id=126)
MABT5102A, (see,
http ://www. alzforum. org/drg/drc/detail. asp? id=121) GSK933766A (see,
Cynthia A. Lemere
& Eliezer Masliah Nature Reviews Neurology 6, 108-119 (February 2010)), IN-N01
(Yamaguchi et al., US Patent No. 7807157), 1C3 (Yamaguchi et al., U.S.
Application No.
12/888,661), 1A-10 (Yamaguchi et al., US Patent No. 7807157) and RN6G (Ding et
al.,
2011, Proc. Natl. Acad. Sci. 108(28) E279-E287).
[0076] Other non-limiting, specific examples of an amyloid beta targeted
antibody is
an exogenous free-end specific antibody which is targeted to a free N-terminus
of amyloid 13
peptide or a free C-terminus of amyloid 13 peptide A131-40. In another
embodiment, an
antibody is targeted to a C-terminus truncated amyloid 13 peptide fragment. In
yet another
embodiment, the antibody is targeted to a free N-terminus of amyloid 13
peptide or a free C-
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terminus of amyloid 13 peptide AP1-40. In another embodiment, the antibody is
free-end
specific and is targeted to the free C-terminus of the amyloid 13-peptide A 13
1-39, A13 1-40,
A13 1-41, or A13 1-43. See, U.S. Patent Application Publication No.
2003/0073655 by Chain.
In a specific embodiment, the antibody may be produced by hybridoma clone
82E1, which
was deposited under the terms of the Budapest Treaty with the International
Patent Organism
Depository, National Institute of Advanced Industrial Science and Technology,
AIST Tsu-
kuba Central 6, 1-1, Higashi 1-chome Tsukuba-shi, Ibaraki-ken 305-8566, Japan
on Feb. 3,
2010 and assigned accession no. FERM BP-11228. See, U.S. Pat. No. 7,807,157,
where that
antibody is characterized in detail.
[0077] The present invention also encompasses antibodies that are targeted to
tau.
Any region of the tau polypeptide may be a suitable target of the antibody. In
a specific
embodiment, an antibody may selectively recognize a phosphorylated form of
tau. In another
embodiment, an antibody may recognize an abnormal tau protein, such as a
truncated form of
tau or a phosphorylated abnormal tau protein, or may recognize a free end of a
truncated
abnormal tau protein. Non-limiting examples of antibodies that target tau,
include, for
example, PHF1 (which recognizes tau with phosphorylated serines 396 and 404
(see,
Boutajangout et al. (2011) J. Neurochem; 1471-4159)); MC1 (a conformation
dependent
antibody that recognizes an early pathological tau conformation) (see, Chai et
al. (2011) J
Biolo Chem; 286:34457-34467), anti-tau (421/422) (see, Garcia-Sierra et al.
(2003) J.
Alzheimer' Disease 5(2) 65-77; Tau C3 (Signet, MBL, Invitrogen); tau dimer and
oligomer
selective TOC-1 (Patterson et al., J. Biol. Chem. 286: 23063-23076); TOMA
antibodies, e.g.,
T2286 (International Patent Application WO/2011/026031); Anti-tau Glu 391,
FITC-tagged
halpha-syn antibody (see, Masliah et al (2005) Neuron; 46: 857-68); TauC3
(which
recognizes tau when truncated at Asp421) (Gamblin et al., Proc Natl Acad Sci U
S A. 2003
August 19; 100(17): 10032-10037); 12E8 (which recognizes tau phosphorylated at
5er262)
(Seubert P., et al., J Biol Chem. 270:18917, 1995; Letersky et al,Biochem. J.,
1996); 1E1/A6
(deSilva R, Lashley T, et al. Neuropath & Applied Neurobio 29 (3):288, 2003);
8E6/C11
(deSilva R, Lashley T, et al. Neuropath & Applied Neurobio 29 (3):288, 2003);
CP27
(Vingtdeux et al., Acta Neuropathol. 2011 Mar;121(3):337-49; Herskovits et
al., Neurobiol
Dis. 2006 Aug;23(2):398-408); CP9 (Roberson et al., Science. 2007 May
4;316(5825):750-
4); DA9 (Zempe et al., J Neurosci. 2010 Sep 8;30(36):11938-50); RTA-1
(Taniguchi T,
Sumida M, et al., FEBS Lett. 579(6):1399-404); RTA-2 (Taniguchi T, Sumida M,
et
al.,FEBS Lett. 579(6):1399-404); CP13 (which detects tau phosphorylated at
5er202); MC6
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(which recognizes tau phosphorylated at Ser 235) (Jicha et al., J. Neurochem.
69, 2087-
2095 (1997)); and 2E12 (highly specific for tau phosphorylated at Thr231)
(Vingtdeux et al.,
Acta Neuropathol. 2011 Mar;121(3):337-49). See also, U.S. Patent Application
No.
61/438,083 by Chain, which describes the structure and sequence of tau and
describes
exemplary antibodies that can be used to target tau in an ADC according to the
present
invention.
[0078] Another target of an antibody of the invention is pathogenic prion
PrPSc. Like
amyloid fibrils, abnormal protein folding into 13-sheet structures to form
amyloid-like
deposits is also widely believed to be the cause of prion-related
encephalophathies, such as
Creutzfeldt-Jakob disease (CJD) and Gerstmann-Straussler-Scheinker disease
(GSS) in
humans, scrapie in sheep and goats, and spongiform encephalopathy in cattle.
The cellular
prion protein (PrPc) is a sialoglycoprotein encoded by a gene that in humans
is located on
chromosome 20. The PrP gene is expressed in neural and non-neural tissues, the
highest
concentration of mRNA being in neurons. The translation product of PrP gene
consists of 253
amino acids in humans, 254 in hamster and mice or 256 amino acids in sheep and
undergoes
several post-translational modifications. In hamsters, a signal peptide of 22
amino acids is
cleaved at the N-terminus, 23 amino acids are removed from the C-terminus on
addition of a
glycosyl phosphatidylinositol (GPI) anchor, and asparagine-linked
oligosaccharides are
attached to residues 181 and 197 in a loop formed by a disulfide bond. In
prion-related
encephalopathies, PrPc is converted into an altered form designated PrPSc,
that is
distinguishable from PrPc in that PrPSc (1) aggregates; (2) is proteinase K
resistant in that
only the N-terminal 67 amino acids are removed by proteinase K digestion under
conditions
in which PrPSc is completely degraded; and (3) has an alteration in protein
conformation
from a-helical for PrPSc to an altered form.
[0079] Any antibody that recognizes pathogenic prion (e.g., misfolded prion
protein,
PrPSc) is contemplated for use in an ADC of the invention. Further, any region
of a
pathogenic prion may be a suitable target of the antibody. A specific example
is ICSM 18
(see, Antonyuk et al. (2009) Proc. Natl Acad Sci USA; 106(8): 2554-2558).
[0080] Anti-a-synuclein antibody therapy for the treatment of Parkinson's
disease is
reviewed in Southwell and Patterson, supra. Anti- a-synuclein antibodies are
contemplated
for use in the present invention. Examples are LB509 which detects C-terminal
alpha
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synuclein (Baba etal., American Journal of Pathology, Vol. 152, No. 4, April
1998), Syn211
and Syn208 (Giasson etal., Neuron. 2002 May 16;34(4):521-33) and 11A5, which
detects a-
synuclein phosphorylated at Serine 129 (Paleologou et al., Neurosci. 2010 Mar
3;30(9):3184-98).
[0081] Any antibody that recognizes pathogenic huntingtin protein, for example
oligomers or cleavage products, is also contemplated for use in an ADC for
this invention.
Further, any region or posttranslational modification of a huntingtin protein
may be suitable
as a target of the antibody. Specific examples are MW1, which binds to the
expanded polyQ
repeat form of Htt, displaying no detectable binding to normal Htt (Ko et al.,
Brain Research
Bulletin Volume 56, Issues 3-4, 1 November 2001, Pages 319-329), EP867Y which
is
specific to the apopain cleavage site of human Huntingtin (Cho et al.,
Neuroscience. 2009
Nov 10; 163(4):1128-34), and MAB2166 (Kaltenback, L. et al. (2007). PloS
Genetics
3:0689-0708).
[0082] Antibodies of the present invention may also recognize al-
antichymotrypsin
(e.g., alpha- 1-Antichymotrypsin (ACT), Verdana Cat No 760-2604; Isaacson P,
et al., (1979)
Lancet. 2:964-965; Palmer PE, et al., (1974) Am J Clin Pathol 62:350-354;
Palmer PE, et al.,
(1980) Cancer 45:1424-1431), apoE (ApoE4 Antibody (4E4) Novus Biologicals Cat
No.
NBP1-49529; Santa Cruz Biotechnology, Santa Cruz, CA) and Creb Binding
Proteins (e.g.,
CBP (C-1): Cat No sc-7300).
[0083] In certain embodiments, an antibody according to the present invention
may
be an IgG4 or IgG2 antibody.
[0084] Antibodies according to the present invention may also be chimeric or
humanized. Publications such as EP0125023, EP0239400, EP045126, W094/20632,
Protein
Eng Des Se!. 2004 May; 17(5):481-489. Epub 2004 Aug. 17; Ann Oncol. 1998 May;
9(5):527-34; and Proc Nat! Acad Sci USA. 1992 May 15; 89(10):4285-9, and the
like can be
referred to for preparation of chimeric antibody and humanized antibody.
Briefly, to create a
humanized antibody, the murine CDRs can be inserted into a human framework
using
methods known in the art. See, e.g., U.S. Patent No. 5,225,539 to Winter, and
U.S. Patent
Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al. Human
antibodies can be
generated by immunizing transgenic or transchromosomic mice in which the
endogenous
mouse immunoglobulin genes have been inactivated and exogenous human
immunoglobulin
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genes have been introduced. Such mice are known in the art (see e.g., U.S.
Patent Nos.
5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016;
5,814,318;
5,874,299; and 5,770,429; all to Lonberg and Kay; U.S. Patent Nos. 5,939,598;
6,075,181;
6,114,598; 6,150,584 and 6,162,963 to Kucherlapati et al; and PCT Publication
WO
02/43478 to Ishida et al.) Human antibodies can also be prepared using phage
display
methods for screening libraries of human immunoglobulin genes. Such phage
display
methods for isolating human antibodies also are known in the art (see e.g.,
U.S. Patent Nos.
5,223,409; 5,403,484; and 5,571,698 to Ladner et al; U.S. Patent Nos.
5,427,908 and
5,580,717 to Dower et al; U.S. Patent Nos. 5,969,108 and 6,172,197 to
McCafferty et al; and
U.S. Patent Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and
6,593,081 to
Griffiths et al.
[0085] Also contemplated for use herein are camel antibodies. Camels generate
functional antibodies consisting of only two heavy chains. These differ from
those of
conventional antibodies in that they lack the CH1 domain. Biophysical studies
have revealed
that camel antibodies have a number of unique features when compared with
those of
conventional antibody molecules, notably their smaller size, greater
solubility and higher
stability See, Tayebi et al. (2010) Journal of General Virology, 91, 2121-
2131. Camel
antibodies are capable of binding to intracellular targets. Non-limiting of
prion specific camel
antibodies are described in Tayebi et al, supra, however, the skilled artisan
that camel
antibodies may be directed against any of the other targets described herein
(e.g., amyloid
beta, tau, apoE, etc.).
[0086] Antibodies of the invention may contain one or more amino acid
mutations,
such as, but not limited to, mutations in the framework regions, or even
within the
complementarity determining regions (CDRs). Preferably, a mutation in the CDR
does not
alter, or enhances, binding of the antibody to its target, compared to the
unmutated antibody.
[0087] Another type of framework modification involves mutating one or more
residues within the framework region or even within one or more CDRs, to
remove T cell
epitopes to thereby reduce the potential immunogenicity of the antibody. This
approach is
also referred to as "deimmunization" and is described in further detail in
U.S. Patent
Publication No. 2003/0153043 by Can et al.
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[0088] In addition or in alternative to modifications made within the
framework or
CDRs, antibodies of the invention may be engineered to include modifications
within the Fc
region, typically to alter one or more functional properties of the antibody,
such as serum
half-life, complement fixation, Fc receptor binding, and/or antigen-dependent
cellular
cytotoxicity. Furthermore, an antibody of the invention may be chemically
modified (e.g.,
one or more chemical moieties can be attached to the antibody) or be modified
to alter its
glycosylation, again to alter one or more functional properties of the
antibody. Each of these
embodiments is described in further detail below.
[0089] In one embodiment, the hinge region of CH1 is modified such that the
number
of cysteine residues in the hinge region is altered, e.g., increased or
decreased. This approach
is described further in U.S. Pat. No. 5,677,425 by Bodmer et al. The number of
cysteine
residues in the hinge region of CH1 is altered to, for example, facilitate
assembly of the light
and heavy chains or to increase or decrease the stability of the antibody.
[0090] In another embodiment, the Fc hinge region of an antibody is mutated to
decrease the biological half-life of the antibody. More specifically, one or
more amino acid
mutations are introduced into the CH2-CH3 domain interface region of the Fc-
hinge fragment
such that the antibody has impaired Staphylococcyl protein A (SpA) binding
relative to
native Fc-hinge domain SpA binding. This approach is described in further
detail in U.S. Pat.
No. 6,165,745 by Ward et al.
[0091] In another embodiment, the antibody is modified to increase its
biological half
life. Various approaches are possible. For example, to increase the biological
half life, the
antibody can be altered within the CH1 or CL region to contain a salvage
receptor binding
epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as
described in
U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.
[0092] In still another embodiment, the C-terminal end of an antibody of the
present
invention is modified by the introduction of a cysteine residue as is
described in detail in WO
2009/026274. Such modifications include, but are not limited to, the
replacement of an
existing amino acid residue at or near the C-terminus of a full-length heavy
chain sequence,
as well as the introduction of a cysteine-containing extension to the c-
terminus of a full-
length heavy chain sequence. In certain embodiments, the cysteine-containing
extension
comprises the sequence alanine-alanine-cysteine (from N-terminal to C-
terminal).
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[0093] In certain embodiments the presence of such C-terminal cysteine
modifications provide a location for conjugation of a partner molecule, such
as a therapeutic
agent or a marker molecule. In particular, the presence of a reactive thiol
group, due to the C-
terminal cysteine modification, can be used to conjugate a partner molecule
employing the
disulfide linkers described in detail below. Conjugation of the antibody to a
partner molecule
in this manner allows for increased control over the specific site of
attachment. Furthermore,
by introducing the site of attachment at or near the C-terminus, conjugation
can be optimized
such that it reduces or eliminates interference with the antibody's functional
properties, and
allows for simplified analysis and quality control of conjugate preparations.
[0094] In still another embodiment, the glycosylation of an antibody is
modified. For
example, an aglycoslated antibody can be made (i.e., the antibody lacks
glycosylation).
Glycosylation can be altered to, for example, increase the affinity of the
antibody for antigen.
Such carbohydrate modifications can be accomplished by, for example, altering
one or more
sites of glycosylation within the antibody sequence. For example, one or more
amino acid
substitutions can be made that result in elimination of one or more variable
region framework
glycosylation sites to thereby eliminate glycosylation at that site. Such
aglycosylation may
increase the affinity of the antibody for antigen. Such an approach is
described in further
detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 to Co et al., Additional
approaches for
altering glycosylation are described in further detail in U.S. Pat. No.
7,214,775 to Hanai et
al., U.S. Pat. No. 6,737,056 to Presta, U.S. Pub No. 20070020260 to Presta,
PCT Publication
No. WO/2007/084926 to Dickey et al., PCT Publication No. WO/2006/089294 to Zhu
et al.,
and PCT Publication No. WO/2007/055916 to Ravetch et al.
[0095] Additionally or alternatively, an antibody can be made that has an
altered type
of glycosylation, such as a hypofucosylated antibody having reduced amounts of
fucosyl
residues or an antibody having increased bisecting GlcNac structures. Such
altered
glycosylation patterns have been demonstrated to increase the ADCC ability of
antibodies.
Such carbohydrate modifications can be accomplished by, for example,
expressing the
antibody in a host cell with altered glycosylation machinery. Cells with
altered glycosylation
machinery have been described in the art and can be used as host cells in
which to express
recombinant antibodies of the invention to thereby produce an antibody with
altered
glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the
fucosyltransferase gene, FUT8 (alpha (1,6) fucosyltransferase), such that
antibodies
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expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their
carbohydrates. The
Ms704, Ms705, and Ms709 FUT8-/- cell lines were created by the targeted
disruption of the
FUT8 gene in CHO/DG44 cells using two replacement vectors (see U.S. Patent
Publication
No. 20040110704 by Yamane et al. and Yamane-Ohnuki et al. (2004) Biotechnol
Bioeng
87:614-22). As another example, EP 1,176,195 by Hanai et al. describes a cell
line with a
functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such
that antibodies
expressed in such a cell line exhibit hypofucosylation by reducing or
eliminating the alpha
1,6 bond-related enzyme. Hanai et al. also describe cell lines which have a
low enzyme
activity for adding fucose to the N-acetylglucosamine that binds to the Fc
region of the
antibody or does not have the enzyme activity, for example the rat myeloma
cell line YB2/0
(ATCC CRL 1662). PCT Publication WO 03/035835 by Presta describes a variant
CHO cell
line, Lec13 cells, with reduced ability to attach fucose to Asn(297)-linked
carbohydrates, also
resulting in hypofucosylation of antibodies expressed in that host cell (see
also Shields, R. L.
et al. (2002)J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 by
Umana et
al. describes cell lines engineered to express glycoprotein-modifying glycosyl
transferases
(e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that
antibodies expressed
in the engineered cell lines exhibit increased bisecting GlcNac structures
which results in
increased ADCC activity of the antibodies (see also Umana et al. (1999) Nat.
Biotech.
17:176-180). Alternatively, the fucose residues of the antibody may be cleaved
off using a
fucosidase enzyme. For example, the fucosidase alpha-L-fucosidase removes
fucosyl residues
from antibodies (Tarentino, A. L. et al. (1975) Biochem. 14:5516-23).
[0096] Additionally or alternatively, an antibody can be made that has an
altered type
of glycosylation, wherein that alteration relates to the level of sialyation
of the antibody. Such
alterations are described in PCT Publication No. WO/2007/084926 to Dickey et
al, and PCT
Publication No. WO/2007/055916 to Ravetch et al. For example, one may employ
an
enzymatic reaction with sialidase, such as, for example, Arthrobacter
ureafacens sialidase.
The conditions of such a reaction are generally described in the U.S. Pat. No.
5,831,077.
Other non-limiting examples of suitable enzymes are neuraminidase and N-
Glycosidase F, as
described in Schloemer et al., J. Virology, 15(4), 882-893 (1975) and in
Leibiger et al.,
Biochem J., 338, 529-538 (1999), respectively. Desialylated antibodies may be
further
purified by using affinity chromatography.
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[0097] Alternatively, one may employ methods to increase the level of
sialyation,
such as by employing sialytransferase enzymes. Conditions of such a reaction
are generally
described in Basset et al., Scandinavian Journal of Immunology, 51(3), 307-
311(2000).
[0098] Another modification of the antibodies herein that is contemplated by
the
invention is pegylation. An antibody can be pegylated to, for example,
increase the biological
(e.g., serum) half life of the antibody. To pegylate an antibody, the
antibody, or fragment
thereof, typically is reacted with polyethylene glycol (PEG), such as a
reactive ester or
aldehyde derivative of PEG, under conditions in which one or more PEG groups
become
attached to the antibody or antibody fragment. Preferably, the pegylation is
carried out via an
acylation reaction or an alkylation reaction with a reactive PEG molecule (or
an analogous
reactive water-soluble polymer). As used herein, the term "polyethylene
glycol" is intended
to encompass any of the forms of PEG that have been used to deriyatize other
proteins, such
as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-
maleimide.
In certain embodiments, the antibody to be pegylated is an aglycosylated
antibody. Methods
for pegylating proteins are known in the art and can be applied to the
antibodies of the
invention. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384
by Ishikawa
et al.
(B) Partner Molecules
[0099] The ADC-containing compositions provided herein comprise an antibody
conjugated to a partner molecule. In certain embodiments, the partner molecule
is a small
molecule. In particular, the small molecule is a cytoprotectiye agent, such
as, but not limited
to, an antioxidant, an antifibrillogenic molecule, or an insulin sensitizing
molecule. Non-
limiting examples of antioxidants according to the present invention include
melatonin, an
indole amine, an indole acid (such as indole-3-propionic acid, and other
indole acids such as
pyruyic and acetic butyric), vitamin E, vitamin C, lipoic acid, uric acid,
circumin,
glutathione, a polyphenol, a flayonoid, an anthraquinone methylthioninium
chloride,
dimebone, a rhodanine-based compounds, an insulin sensitizer e.g. pioglitazone
and
rosiglitazone, an 8-hydroxyquinolone derivative e.g. PBT2, PBT434,
penicillamine, trientine,
a tetracycline, (N-
(pyridin-2-ylmethyl)aniline), N1,N1-dimethyl-N4-(pyridin-2-
ylmethyl)benzene-1,4-diamine, 2,6-diaminopyridine, AZD-103, cyclohexane-
1,2,3,4,5,6-
hexol, myo-inositol, scyllo-inositol, indole-3-propionic acid, (N-(pyridin-2-
ylmethyl)aniline),
methylene blue, TRx0014, and NO scavengers.
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[00100] Examples
of partner molecules include melatonin and melatonin
analogs that retain the antioxidant properties of melatonin, and preferably,
although not
necessarily, the ability to interact with the high affinity copper binding
site in AP and to
inhibit fibril formation.
[00101] Analogs
of melatonin include compounds that interact with
melatonergic systems, for example, compounds that interact with the melatonin
receptor.
Many examples of such compounds are known in the art. See, for example, U.S.
Pat. Nos.
5,449,690, 5,464,872, 5,470,846, 5,541,228, 5,552,418, 5,552,428, 5,554,642,
5,580,878, and
5,591,775. Melatonin analogs can readily be assayed to ensure that the
antioxidant function
of melatonin is retained, using the methodology disclosed herein, such as
assays for cell
viability, lipid peroxidation, intracellular Ca2+, and oxygen free-radicals.
The prevention of
other cytotoxic effects of AP on cells can readily be observed
microscopically, such as the
prevention of membrane blebbing, cell retraction, abnormal distribution of
chromatin, and
karyorrhexis. As indicated above, the cytotoxic or cell killing effects of AP
include, for
example, decreased cell viability (i.e. cell death), increased lipid
peroxidation (an indicator of
increased oxygen free-radicals), increased intracellular Ca2+, levels, diffuse
membrane
blebbing, cell retraction, abnormal distribution of chromatin towards the
nuclear membrane,
and karyorrhexis. The cytotoxic effects of AP are most readily seen in
neuronal cells
(including cells of the central and peripheral nervous systems), and occur in
human subjects
afflicted with AD. It may also be determined whether the analogue maintains
the ability to
interact with the high affinity copper binding site in amyloid beta.
[00102] Among
possible neuroprotective agents, frontline candidates are those
that prevent protein aggregation, inhibit accumulation of reactive oxygen
species or prevent
other processes before irreversible damage to nerve cells has occurred.
Neuroprotective
approaches to the treatment of theses diseases can include free radical
scavengers, especially
hydroxyl radical scavengers, nitrous oxide scavengers, selective metal
chelators, metal
attenuating compounds, electron transfer stimulators, and anti-inflammatory
compounds.
[00103] The
endogenous hormone melatonin holds promise as a
neuroprotective compound for the treatment of neurodegenerative disease and
its age related
decline might also contribute to increased levels of oxidative stress in the
elderly. Melatonin
is a potent antioxidant consuming 4 moles of hydroxyl radical per mole of
melatonin. The
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compound has also been shown to bind to amyloid beta and to inhibit the
progressive
formation of 13sheets and fibrils. Melatonin also has multiple actions as a
regulator of
antioxidant and prooxidant enzymes, radical scavenger and antagonist of
mitochondrial
radical formation. Moreover, melatonin is incapable of forming damaging pro-
oxidant
intermediates upon metabolism. The ability of melatonin and its kynuramine
metabolites to
interact directly with the electron transport chain by increasing the electron
flow and reducing
electron leakage are unique features by which melatonin is able to increase
the survival of
neurons under enhanced oxidative stress. Moreover, antifibrillogenic actions
of melatonin
have been demonstrated in vitro, also in the presence of profibrillogenic
apoE4 or apoE3, and
in vivo, in a transgenic mouse model. Amyloid beta toxicity is prevented by
melatonin and
analogues such as indole-3-propionic acid in vitro models. Melatonin treatment
has been also
demonstrated to be beneficial in animal models of AD, PD and HD and to protect
retinal
epithelial cells in models of AMD. However, melatonin has had modest benefit
affecting
disease progress in human clinical trials of AD except by improving sleep
disturbances based
on its receptor-mediated regulation of circadian rhythms at low doses of the
hormone. At
higher doses, such as for neuroprotection, melatonin may be expected to result
in adverse off-
target effects. According to the Mayo Clinic
(http://www.mayoclinic.comihealthimelatonin-
side-effects/AN01717), melatonin side effects may include daytime sleepiness,
dizziness,
headaches, abdominal discomfort, confusion, sleepwalking, and nightmares.
Melatonin may
also interact with various medications, including blood-thinning medications
(anticoagulants), immunosuppressants and diabetes medications. It would be
desirable to
prevent some of the side effect of melatonin, such as by preventing binding of
melatonin to
its receptor following administration to a subject.
[00104] A major
limitation of current melatonin based therapies for the
treatment of proteinopathies is that it has an extremely short half life in
the blood and in the
eye which leads to poor bioavailability and the need for potentially harmful
high doses
administered chronically over many years. It would be desirable to increase
the half-life of
melatonin and thus widen the therapeutic window.
[00105] In human
antioxidant studies, Vitamin E (a-tocopherol), a potent chain
breaking antioxidant, is one of the most extensively studies antioxidant
agents. Data from
cross-sectional and longitudinal studies assessing the relationship between
Vitamin E
consumption and AD risk have led to conflicting resulted while high levels of
Vitamin
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consumption over time in the general population has been associated with
increased
morbidity. The most promising clinical trial to date was conducted with
Vitamin E
supplementation of 2000 IU per day for moderate-stage AD patients which led to
a small but
significant delay in reaching the end points of institutionalization, loss of
major activities of
daily living, or death, but did not delay loss of cognitive performance.
[00106] A major
limitation of Vitamin E as a potential therapy for AD or other
proteinopathies affecting the brain is that even a massive increase in vitamin
E intake does
not result in an increase of this vitamin's concentration in the brain because
of its poor BBB
permeation and tight regulation within the brain. In contrast, liver and
muscle are well-
receptive of rising Vitamin E supplies. It would be desirable to increase the
bioavailability of
Vitamin E in the brain without altering its antioxidant activity and deliver
concentrated
localized concentrations of the drug to the sites of amyloidosis, preferably
together with an
agent that promotes amyloid clearance.
[00107] There is
substantial in-vitro data indicating that Curcumin has
antioxidant, anti-inflammatory, and anti-amyloid activity. In addition,
studies in AD animal
models of indicate a direct effect of Curcumin in decreasing the amyloid
pathology of AD.
As the widespread use of Curcumin as a food additive and relatively small
short-term studies
in humans suggest safety, Curcumin is a promising agent in the treatment
and/or prevention
of AD.
[00108] Two
major limitations of Curcumin as a therapy for AD are first, that it
has very poor penetration into the brain and secondly, that it is rapidly
eliminated from the
body. It would be desirable to increase the penetration of Curcumin into the
brain, increase
brain retention, deliver high, localized concentrations to sites of oxidative
stress caused by
amyloid proteins and reduce its elimination from the blood, preferably
together with an agent
that promotes amyloid clearance.
[00109] Cu2+ and
Zn2+ promote aggregation of Ap and chelation of these
metals renders AP aggregates less compact and less resistant turnover.
Derivatives of 8-
Hydroxyquinoline are being developed as potential therapeutics for
proteinopathies based on
the metal attenuating properties. PBT2 is a copper/zinc ionophore that rapidly
restores
cognition in AD mouse models and also shown beneficial effects in HD models. A
recent
Phase Ha double-blind, randomized, placebo-controlled trial found that the 250
mg dose of
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PBT2 was well-tolerated, significantly lowered cerebrospinal fluid (CSF)
levels of amyloid-
beta42, and significantly improved executive function on a Neuro-psychological
Test Battery
(NTB) within 12 weeks of treatment in patients with AD. PBT434 has been shown
that it is
able to impede the iron-induced oxidative damage and neurotoxic cascade that
kills the
substantia nigra in PD. Other metal chelating compounds of interest include
trientine,
penicillamine, 2,6-diaminopyridine.
[00110] A
limitation of metal binding molecules is the possibility of interfering
with non-pathogenic metal dependent enzymes and other physiological processes.
It would be
desirable to be able to deliver high, localized concentrations of PBT2 and
PBT434 in the
vicinity of amyloidogenic metal bound proteins such as AP, alpha synuclein and
huntingtin,
preferably together with an agent that promotes amyloid clearance.
[00111]
Rhodanine-based compounds are frequently employed in medicinal
chemistry with no apparent side effects and the core has been investigated for
tau aggregation
inhibition via the synthesis of a focused library. Rhodanine heterocycle was
the most effect
compound with an IC50 of 0.81.iM representing the assembly inhibiting half
maximal
concentration measured in vitro and an DC50 of 0.11AM in relation to
disassembling-inducing
activity. The rhodanine heterocycle appeared to contribute the main activity
in each case.
Besides the central rhodanine core, hydrogen bond acceptors in the form of a
nitro group,
carboxylic acids, phenols, sulfonates/sulfonamides are required in line with
observations
from other known amyloid inhibitors. Noteworthy, the total length of the
molecule proved to
be of importance. Variations of the length of the linker between the
carboxylic acid and the
rhodanine core revealed that increasing the distance up to two carbons bonds
resulted in an
appreciable increase in the compound's inhibitory potency indicating the
optimal positioning
of the inhibitor toward its bindings site. An example of a rhodanine compound
suitable for
this invention is pioglitazone. The mechanism for development of AD has been
linked to both
inflammation and decreased insulin sensitivity. Because of this, pioglitazone
has been
evaluated as potential treatment for AD because of its insulin-sensitizing and
anti-
inflammatory effects.
[00112] A
significant limitation of rhodanine-based compounds as therapeutic
compounds for treating AD and other tauopathies is that relative potencies in
vitro are not
well correlated in vivo because of various ADME parameters (adsorption,
distribution,
metabolism and excretion indicating that molecules need to be carefully
optimized. It would
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be desirable to select compounds based on potency determine from in vitro
assays without
need for further optimization.
[00113] Numerous
polyphenols show inhibitory activity in a variety of
amyloids such as alpha synuclein, IAPP, A13, PrPsc. Mycetin has been reported
as tau
aggregation inhibitors with a 1.2 1..EM IC50 and the in vivo data indicated
with the elongation
phase of the fibril assembly.
[00114] A major limitation of dietary polyphenols is due to
lack of
bioavailability including poor uptake across the intestine followed by
extensive metabolism
on reaching the plasma through methylation, sulfation and glucuronidation.
Moreover, while
penetration across the blood brain barrier is thought to be poor, high brain
concentrations of
polyphenols pose significant safety concerns with increasing evidence of acute
polyphenol
toxicity at high doses. It would be desirable to have a chaperone to improve
the
bioavailability of polyphenols while concentrating their neuroprotective
function to sites of
amyloidos is .
[00115] Solid-
state nuclear magnetic resonance (ssNMR) may be used to
identify key residues within amyloidogenic protein sequences that may be
targeted to inhibit
the aggregation of the host protein. For a-synuclein, the major protein
component of Lewy
bodies associated with Parkinson's disease, combination of ssNMR and
biochemical data was
used to identify the key region for self-aggregation of the protein as
residues 77-82
(VAQKTV) which led to the design of a new peptide derived from residues 77 to
82 of a-
synuclein with an N-methyl group at the C-terminal residue, which was able to
disrupt the
aggregation of a-synuclein.
[00116] A major
limitation of using short peptides as therapeutic for the
treatment of proteinopathies is that they are rapidly eliminated from the
body. It would be
desirable to increase the bioavailability of short peptide inhibitors of
protein aggregation and
increase retention in the brain
[00117] LMTXTm,
is a second-generation tau aggregation inhibitor containing
the active ingredient, methylthioninium (MT). The LMTXTm family also has
activity against
synuclein aggregation. LMTX activity could be synergistic if applied with
other therapies for
example an antibody against either tau protein.
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[00118] Suitable
assays for determining whether a melatonin analogue
maintains one or more properties or functions of melatonin (e.g., an ability
to interact with
amyloid beta peptide and/or to inhibit fibril formation) are described in
detail in Pappolla et
al. (1997) J Biol Chem 273(13):7185-7188. See, also, Cheng et al. (2005) Anal.
Chem. 2005,
77, 7012-7015, which describes a mass spectrometry-based screening assay for
identifying
compounds that inhibit the aggregation of Ail
[00119]
Oxidative damage has been suggested to be the primary cause of aging
and age-associated neurodegenerative diseases like AD, PD, and HD. This
concept is based
on the free radical hypothesis of aging as proposed by Harman. See, Harman D:
(1956). J
Gerontol; 11:298-300. There is compelling evidence for a decisive
participation of severe
oxidative stress in the development of neuropathology seen in AD.
Immunohistochemical
studies confirmed findings demonstrating increased levels of lipid
peroxidation in vitro
observed in autopsy samples of brains afflicted by AD. Because of its high
rate of oxygen
consumption and its high content of polyunsaturated fatty acids, the brain
exhibits increased
vulnerability to oxidative stress. Elevated lipid peroxidation, as found in
the brains of AD
patients, not only reveals oxidative stress, but also exerts secondary effects
on protein
modification, oxidation and conformation.
[00120]
Increased protein and DNA oxidation also occurs in AD.
Measurements of protein carbonyl, 3,3'-dityrosine and 3-nitrotyrosine in post
mortem brain
samples from AD patients have shown increased oxidative and nitrosative
protein
modification in the hippocampal and neocortical regions, but not in the
cerebellum. Free
radical attack on DNA results in strand breaks, DNA-protein cross linkage, and
base
modification. Double- and single-strand breaks were elevated in AD cortex and
hippocampus, but this has to be largely attributed to apoptotic fragmentation.
Enhanced
oxidative DNA modification is, however, also demonstrable, mostly as 8-hydroxy-
2'-
deoxyguanosine (8-0HdG), a product primarily formed by attack of hydroxyl
radicals, but
other modified bases such as 8-OH-adenine have also been demonstrated.
[00121]
Augmented free radical damage to lipids, proteins and nucleic acids
has been reported for the substantia nigra of parkinsonian patients.
Therefore, numerous
compounds with antioxidant properties have been suggested for treatment of AD
and other
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neurodegenerative diseases. Among these substances, melatonin is unique for
several
reasons: it is a natural compound synthesized in the pineal gland and other
body tissues; it
can be released by the pineal gland via the pineal recess into the
cerebrospinal fluid (CSF), in
much higher concentrations than into the circulation; its production decreases
with the
advancement of age, a fact which has been suggested to be one of the major
causes of age-
associated neurodegenerative diseases. For a comprehensive review of
melatonin, see
Srinivasan et al. (2006) Behavioral and Brain Functions, 2:15.
[00122] A
partner molecule, such as, e.g., an antioxidant, may also be
modified, e.g., to further increase bioavailability. For example, an
antioxidant may be
chemically modified with a fluorinated amphiphilic carrier to increase
bioavailability and/or
half-life in vivo. See, Ortial et al. (2006) J Med Chem. 49(9):2812-20.
[00123] A
partner molecule may also be labeled with a marker. Labels are
useful for enabling, e.g., in vivo imaging of a site targeted by an ADC of the
invention. (e.g.,
amyloid deposit and/or neurofibrillary tangle). For example, and without
limitation, a marker
can be any label that generates a detectable signal, such as a radiolabel, a
fluorescent label
(e.g., GFP), or an enzyme that catalyzes a detectable modification to a
substrate (e.g.,
horseradish peroxidase).
[00124] Specific
examples of how partner molecules can be conjugated are
given below: In certain cases, these small molecules (or "payloads") could be
conjugated via
use of non-cleavable linkers through available functional groups, for example,
hydroxyl or
thiol groups, whereas in other cases, the payload may require modification
before it can be
conjugated either to a cleavable or non-cleavable linker. For example, the
phenolic OH
group is available as a reactive group in tocopherol and curcumin and could be
used in
combination with a non-cleavable linker. However, it would be important to
determine if the
free radical scavenging properties of tocopherol or curcumin are inhibited in
this way, in
which case it may be preferable to use cleavable linkers as described in WO
2007089149A2
or J. Med. Chem. 2005, 48, 1344-1358. The same is true for derivatives of 8-
Hydroxyquinoline such as PBT2 or PBT434 and with respect to the OH group in
indole-3-
propionic acid. Since both the neurogenesis enhancers P7C3A20 and P7C3 are
active, it may
be preferable to conjugate P7C3, which has an OH group. For penicillamine, one
might use
SMCC linker (e.g. T-DM1) or similar for conjugation via the free thiol group
or use the
primary amine in the context of cleavable linkers. Similarly, one can make use
of the
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primary amines in 2,6-diaminopyridine and its derivatives and similarly with
trientine. In
view of the absence of available free functional groups in molecules such
pioglitazone,
TRx0014 and Ponatinib, for example, these drugs would need to be modified
before they
could be conjugated. Table 1 provides a list of partner molecules of the
invention with
possible antibody combinations and linker types.
Table 1: Example Antibody Conjugates of the Invention
Partner Molecule Partner Molecule Structure Antibody Linker
Melatonin Ei,c; Amyloid beta Non-cleavable
litst¨i 41.. --CH,
0
alpha tocopherol Amyloid beta Non-cleavable
8- alpha Cleavable
Hydroxyquinoline synuclein
"
derivative e.g. ' I
;k.
PBT434
REMBER Tau Cleavable/non-
TRx0014 cleavable
,
Rhodanine H Tau Non-cleavable
derivatives
8
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phenylthiazolyl- Tau Non-cleavable
hydrazide
derivatives
Pioglitazone amyloid beta, Cleavable
tau, alpha
N ,C$
-1" synuclein &
huntingtin
[00125] Antibody-
drug conjugates have more complex and heterogenous
structures than the corresponding antibodies. The selection of the most
appropriate methods
for a specific ADC is heavily dependent on the properties of the linker, the
drug and the
choice of attachment sites (lysines, interchain cysteines, Fc glycans).
Improvements in
analytical techniques such as protein mass spectrometry and capillary
electrophoresis have
significantly increased the quality of information that can be obtained for
use in product and
process characterization and for routine lot release and stability testing
necessary to ensure
that they are optimized for use in pharmacological compositions. Methods and
approaches
are reviewed in Wakankar et al., 2011, mAbs 3:2 161-172 (Landes Bioscience).
(C) Linkers
[00126] In some
embodiments of the invention, an antibody is conjugated
directly to a partner molecule (e.g., cytoprotective agent or other small
molecule) via a
cysteine residue at or near the C-terminus of a heavy chain of the antibody.
The cysteine
residue may be naturally occurring in the heavy chain or, in certain
embodiments, the
cysteine residue at the C-terminus of the heavy chain is introduced by the
replacement of the
original C-terminal amino acid residue.
[00127] In other
embodiments, a partner molecule is conjugated to an antibody
by a chemical linker (sometimes referred to herein simply as "linker").
[00128] The
ratio of partner molecules attached to an antibody can vary,
depending on factors such as the amount of partner molecule employed during
conjugation
reaction and the experimental conditions. Preferably, the ratio of partner
molecules to
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antibody is between 1 and 20, more preferably between 1 and 10. Those skilled
in the art will
appreciate that, while each individual molecule of antibody Z is conjugated to
an integer
number of partner molecules, a preparation of the conjugate may analyze for a
non-integer
ratio of partner molecules to antibody, reflecting a statistical average.
[00129] Linkers
inherently have shorter half-lives than their antibody
counterparts, and therefore typically, although not necessarily, need to be
modified to
improve their solubility. This can be accomplished, for example, by
conjugating a
polyethylene glycol (PEG), or PEG-like derivative, to the linker. However, it
will be
understood that other modifications are known to those of skill in the art and
can be used in
the ADC described herein.
[00130] In some
embodiments, the linker is a peptidyl linker, depicted herein as
(L4)p-F-(L1)õ. Other linkers include hydrazine and disulfide linkers, depicted
herein as
H-(L1)õ, and (L4)p-J-(L1)õ, respectively. F, H, and J are peptidyl, hydrazine,
and disulfide
moieties, respectively, that are cleavable to release the partner molecule
from the antibody,
while L1 and L4 are linker groups. F, H, J, L1, and L4 are more fully defined
herein below,
along with the subscripts p and m. The preparation and use of these and other
linkers are
described in detail in WO 2005/112919.
[00131] The use
of peptidyl and other linkers in ADC is described in U.S. Pat.
Application Pub. No. 2006/0004081; 2006/0024317; 2006/0247295; U.S. Pat. No.
6,989,452;
U.S. Pat. No. 7,087,600; and U.S. Pat. No. 7,129,261; WO 2007/051081; WO
2007/038658;
WO 2007/059404; and WO 2007/089100. Additional linkers are described in U.S.
Pat. No.
6,214,345; and U.S. Pat. Application Pub. No. 2003/0096743; and 2003/0130189;
in de
Groot et al., J. Med. Chem. 42, 5277 (1999); de Groot et al. J. Org. Chem. 43,
3093 (2000);
de Groot et al., J. Med. Chem. 66, 8815, (2001); WO 02/083180; Carl et al., J.
Med. Chem.
Lett. 24, 479, (1981); Dubowchik et al., Bioorg & Med. Chem. Lett. 8, 3347
(1998).
[00132] In
addition to connecting the antibody and the partner molecule, a
linker can impart stability to the partner molecule, reduce its in vivo
toxicity, or otherwise
favorably affect its pharmacokinetics, bioavailability and/or
pharmacodynamics. It is
generally preferred that the linker is cleaved, releasing the partner
molecule, once the
conjugate is delivered to its site of action. Also preferably, the linkers are
traceless, such that
once cleaved, no trace of the linker's presence remains.
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[00133] In
another embodiment, the linkers are characterized by their ability to
be cleaved at a site in or near a target cell such as at the site of
therapeutic action or marker
activity of the partner molecule. Such cleavage can be enzymatic in nature.
This feature aids
in reducing systemic activation of the partner molecule, reducing toxicity and
systemic side
effects. Preferred cleavable groups for enzymatic cleavage include peptide
bonds, ester
linkages, and disulfide linkages, such as the aforementioned F, H, and J
moieties. In other
embodiments, the linkers are sensitive to pH and are cleaved through changes
in pH. For
example, a cleavable linker may be preferably hydrolysable at a pH of less
than 5.5.
[00134] One
aspect is the ability to control the speed with which the linkers
cleave. Often a linker that cleaves quickly is desired. In some embodiments,
however, a
linker that cleaves more slowly may be preferred. For example, in a sustained
release
formulation or in a formulation with both a quick release and a slow release
component, it
may be useful to provide a linker which cleaves more slowly. WO 2005/112919
discloses
hydrazine linkers that can be designed to cleave at a range of speeds, from
very fast to very
slow.
[00135] Linkers
can serve to stabilize the partner molecule against degradation
while the conjugate is in circulation, before it reaches the target tissue or
cell. This is a
significant benefit since it prolongs the circulation half-life of the partner
molecule. A
preferred partner molecule of the invention, melatonin, for example, has a
particularly short
half-life in blood. In some embodiments, the linker can serve to attenuate the
activity of the
partner molecule so that the conjugate is relatively benign while in
circulation but the partner
molecule has the desired effect, e.g., protects cells from oxidotoxins, after
activation at the
desired site of action. For therapeutic agent conjugates, this feature of the
linker serves to
improve the therapeutic index of the agent.
[00136] In
addition to the cleavable peptide, hydrazine, or disulfide groups F,
H, or J, respectively, one or more linker groups L1 are optionally introduced
between the
partner molecule and F, H, or J, as the case may be. These linker groups L1
may also be
described as spacer groups and contain at least two functional groups.
Depending on the
value of the subscript m (i.e., the number of L1 groups present) and the
location of a particular
group L1, a chemical functionality of a group L1 can bond to a chemical
functionality of the
partner molecule, of F, H or J, as the case may be, or of another linker group
L1 (if more than
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one L1 is present). Examples of suitable chemical functionalities for spacer
groups L1 include
hydroxy, carbonyl, carboxy, amino, ketone, aldehyde, and mercapto groups.
[00137] The
linkers L1 can be a substituted or unsubstituted alkyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl or substituted or
unsubstituted
heteroalkyl group. In one embodiment, the alkyl or aryl groups may comprise
between 1 and
20 carbon atoms. They may also comprise a PEG moiety.
[00138]
Exemplary groups L1 include, for example, 6-aminohexanol, 6-
mercaptohexanol, 10-hydroxydecanoic acid, glycine and other amino acids, 1,6-
hexanediol,
13-alanine, 2-aminoethanol, cysteamine (2-aminoethanethiol), 5-aminopentanoic
acid, 6-
aminohexanoic acid, 3-maleimidobenzoic acid, phthalide, a-substituted
phthalides, the
carbonyl group, aminal esters, nucleic acids, peptides and the like.
[00139] One
function of the groups L1 is to provide spatial separation between
F, H or J, as the case may be, and the partner molecule, lest the latter
interfere (e.g., via steric
or electronic effects) with cleavage chemistry at F, H, or J. The groups L1
also can serve to
introduce additional molecular mass and chemical functionality into conjugate.
Generally, the
additional mass and functionality affects the serum half-life and other
properties of the
conjugate. Thus, through careful selection of spacer groups, conjugates with a
range of serum
half-lives can be produced. Optionally, one or more linkers L1 can be a self-
immolative
group, as described herein below.
[00140] The
subscript m is an integer selected from 0, 1, 2, 3, 4, 5, and 6. When
multiple L1 groups are present, they can be the same or different.
[00141] L4 is a
linker moiety that provides spatial separation between F, H, or
J, as the case may be, and the antibody, lest F, H, or J interfere with the
antigen binding by
the antibody or the antibody interfere with the cleavage chemistry at F, H, or
J. Preferably, L4
imparts increased solubility or decreased aggregation properties to conjugates
utilizing a
linker that contains the moiety or modifies the hydrolysis rate of the
conjugate. As in the case
of L1, L4 optionally is a self immolative group. In one embodiment, L4 is
substituted alkyl,
unsubstituted alkyl, substituted aryl, unsubstituted aryl, substituted
heteroalkyl, or
unsubstituted heteroalkyl, any of which may be straight, branched, or cyclic.
The
substitutions can be, for example, a lower (C1-C6) alkyl, alkoxy, alkylthio,
alkylamino, or
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dialkyl-amino. In certain embodiments, L4 comprises a non-cyclic moiety. In
another
embodiment, L4 comprises a positively or negatively charged amino acid
polymer, such as
polylysine or polyarginine, L4 can comprise a polymer such as a PEG moiety.
Additionally,
L4 can comprise, for example, both a polymer component and a small molecule
moiety.
[00142] In a preferred embodiment, L4 comprises a PEG moiety. The PEG
portion of L4 may be between 1 and 50 units long. Preferably, the PEG will
have 1-12 repeat
units, more preferably 3-12 repeat units, more preferably 2-6 repeat units, or
even more
preferably 3-5 repeat units and most preferably 4 repeat units. L4 may consist
solely of the
PEG moiety, or it may also contain an additional substituted or unsubstituted
alkyl or
heteroalkyl. It is useful to combine PEG as part of the L4 moiety to enhance
the water
solubility of the complex. Additionally, the PEG moiety reduces the degree of
aggregation
that may occur during the conjugation of the drug to the antibody.
[00143] The subscript p is 0 or 1; that is, the presence of L4 is
optional. Where
present, L4 has at least two functional groups, with one functional group
binding to a
chemical functionality in F, H, or J, as the case may be, and the other
functional group
binding to the antibody. Examples of suitable chemical functionalities of
groups L4 include
hydroxy, carbonyl, carboxy, amino, ketone, aldehyde, and mercapto groups. As
antibodies
typically are conjugated via sulfhydryl groups (e.g., from unoxidized cysteine
residues, the
addition of sulfhydryl-containing extensions to lysine residues with
iminothiolane, or the
reduction of disulfide bridges), amino groups (e.g., from lysine residues),
aldehyde groups
(e.g., from oxidation of glycoside side chains), or hydroxyl groups (e.g.,
from serine
residues), preferred chemical functionalities for attachment to the antibody
are those reactive
with the foregoing groups, examples being maleimide, sulfhydryl, aldehyde,
hydrazine,
semicarbazide, and carboxyl groups. The combination of a sulfhydryl group on
the antibody
and a maleimide group on L4 is preferred.
[00144] In some embodiments, L4 comprises:
0 R26' R25,
t T s
R26 R25
R20
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directly attached to the N-terminus of (AA1),. R2 is a member selected from
H, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl, and acyl. Each
R25, R25', K-26,
and
R26' is independently selected from H, substituted or unsubstituted alkyl,
substituted or
unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or
unsubstituted
heteroaryl, and substituted or unsubstituted heterocycloalkyl; and s and t are
independently
integers from 1 to 6. Preferably, R20, R25, R25', x-26
and R26' are hydrophobic. In some
embodiments, R2 is H or alkyl (preferably, unsubstituted lower alkyl). In
some
embodiments, R20, R25, R25', R26
and R26' are independently H or alkyl (preferably,
unsubstituted C1 to C4 alkyl). In some embodiments, R20, R25, R25', R26 and lc-
26'
are all H. In
some embodiments, t is 1 and s is 1 or 2.
[00145] Linker
moieties of the invention also include, e.g., thioether linkers
derived from maleimide derivatives. For example, maleimides are known to react
with
sulfhydryl groups at pH 6.5-7.5 to form stable thioether bonds. In particular,
the compound
SMCC (N-succinimidy1-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate), having
the
structure:
17-^C(1417.$1105
contains a sulfhydryl-reactive maleimide group to form a stable thioether
bond. SMCC also
contains an amine-reactive N-hydroxysuccinimide ester which reacts with
primary amines.
This compound is therefore well suited as a cross-linking agent for direct
conjugation of
antibodies to other functional moieties, such as partner molecules. In some
embodiments, for
example, the ADC is an antifibrillogenic molecule attached by an SMCC linker
that is
extended by a short PEG motif to increase the spatial separation between the
partner
molecule and the antibody backbone. See, Vollhardt and Schore, Organic
Chemistry:
Structure and Function, Fourth Edition, W. H. Freeman & Co. 0 2002 W. H.
Freeman & Co.,
and Sumanas, Inc.
(i) Peptide Linkers (F)
[00146] As
discussed above, the peptidyl linkers of the invention can be
represented by the general formula: ((L4)p-F-(L1)õ, wherein F represents the
portion
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comprising the peptidyl moiety. In one embodiment, the F portion comprises an
optional
additional self-immolative linker L2 and a carbonyl group, corresponding to a
conjugate of
formula (a):
0
X4 ¨ (L4), ¨ (AA1),¨ (L2 ¨ C), ¨ (L1)¨D.
In this embodiment, L1, L4, p, and m are as defined above. X4 is an antibody
and D is a
partner molecule. The subscript o is 0 or 1 and L2, if present, represents a
self-immolative
linker. AA1 represents one or more natural amino acids, and/or unnatural a-
amino acids; c is
an integer from 1 and 20. In some embodiments, c is in the range of 2 to 5 or
c is 2 or 3.
[00147] In
formula (a), (AA1) is linked, at its amino terminus, either directly to
L4 or, when L4 is absent, directly to X4. In some embodiments, when L4 is
present, L4 does
not comprise a carboxylic acyl group directly attached to the N-terminus of
(AA1),.
[00148] In
another embodiment, the F portion comprises an amino group and
an optional spacer group L3 and L1 is absent (i.e., m is 0), corresponding to
a conjugate of
formula (b):
x4 ¨ (L4)p¨ (AA1), ¨ N¨ (L3),¨ D.
In this embodiment, X4, D, L4, (AA1),, and p are as defined above. The
subscript o is 0 or 1.
L3, if present, is a spacer group comprising a primary or secondary amine or a
carboxyl
functional group, and either the amine of L3 forms an amide bond with a
pendant carboxyl
functional group of D or the carboxyl of L3 forms an amide bond with a pendant
amine
functional group of D.
(ii) Self-Immolative Linkers
A self-immolative linker is a bifunctional chemical moiety which is capable of
covalently
linking together two spaced chemical moieties into a normally stable
tripartate molecule,
releasing one of said spaced chemical moieties from the tripartate molecule by
means of
enzymatic cleavage; and following said enzymatic cleavage, spontaneously
cleaving from the
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remainder of the molecule to release the other of said spaced chemical
moieties. In
accordance with the present invention, the self-immolative spacer is
covalently linked at one
of its ends to the peptide moiety and covalently linked at its other end to
the chemically
reactive site of the drug moiety whose derivatization inhibits pharmacological
activity, so as
to space and covalently link together the peptide moiety and the drug moiety
into a tripartate
molecule which is stable and pharmacologically inactive in the absence of the
target enzyme,
but which is enzymatically cleaved by such target enzyme at the bond
covalently linking the
spacer moiety and the peptide moiety to thereby effect release of the peptide
moiety from the
tripartate molecule. Such enzymatic cleavage, in turn, will activate the self-
immolating
character of the spacer moiety and initiate spontaneous cleavage of the bond
covalently
linking the spacer moiety to the drug moiety, to thereby effect release of the
drug in
pharmacologically active form. See, for example, Carl et al., J. Med. Chem.,
24 (3), 479-480
(1981); WO 81/01145; Toki et al., J. Org. Chem. 67, 1866-1872 (2002); WO
2005/112919;
and WO 2007/038658. See, also, U.S. Pat. No. 7,375,078.
[00149] One
particularly preferred self-immolative spacer may be represented
by the formula (c):
K,
n
;
N -/
R24
The aromatic ring of the aminobenzyl group may be substituted with one or more
"K" groups.
A "K" group is a substituent on the aromatic ring that replaces a hydrogen
otherwise attached
to one of the four non-substituted carbons that are part of the ring
structure. The "K" group
may be a single atom, such as a halogen, or may be a multi-atom group, such as
alkyl,
heteroalkyl, amino, intro, hydroxy, alkoxy, haloalkyl, and cyano. Each K is
independently
selected from the group consisting of substituted alkyl, unsubstituted alkyl,
substituted
heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl,
substituted
heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl,
unsubstituted
heterocycloalkyl, halogen, NO2, NR
21R22, NR21c0R22,
0C0NR21R22, 000R21, and OR21,
wherein R21 and R22 are independently selected from the group consisting of H,
substituted
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alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted
heteroalkyl, substituted aryl,
unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl,
substituted
heterocycloalkyl and unsubstituted heterocycloalkyl. Exemplary K substituents
include, but
are not limited to, F, Cl, Br, I, NO2, OH, OCH3, NHCOCH3, N(CH3)2, NHCOCF3 and
methyl. For "K,", i is an integer of 0, 1, 2, 3, or 4. In one preferred
embodiment, i is 0.
[00150] The ether oxygen
atom of the above structure is connected to a
carbonyl group (not shown). The line from the NR24 functionality into the
aromatic ring
indicates that the amine functionality may be bonded to any of the five
carbons that both form
the ring and are not substituted by the ¨CH2-0¨ group. Preferably, the NR24
functionality
of X is covalently bound to the aromatic ring at the para position relative to
the ¨CH2-0¨
group. R24 is a member selected from the group consisting of H, substituted
alkyl,
unsubstituted alkyl, substituted heteroalkyl, and unsubstituted heteroalkyl.
In a specific
embodiment, R24 is hydrogen.
[00151] In one embodiment,
the invention provides a peptide linker of formula
(a) above, wherein F comprises the structure:
0
K, II
(1=\ ____________________ f-C
[ I __________ d
(AA1)c-N, 1/2. '
R24
where R24, (AA1), K, and i are as defined above.
[00152] In another
embodiment, the peptide linker of formula (a) above
comprises a ¨F-(L1)õ- that comprises the structure:
R24 R24 R24
0
K, II
C-1=) OCN
_________________ / I
1 -2,4"<- / R24 R24 R24 0
__ (AA ')-N
I
R24
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where R24, (AA1), K and i are as defined above.
[00153] In some embodiments, a self-immolative spacer L1 or L2
includes
RI7
NJR
(RI9).
where each R12, R18, and R19 is independently selected from H, substituted or
unsubstituted
alkyl, substituted or unsubstituted heteroalkyl and substituted or
unsubstituted aryl, and w is
an integer from 0 to 4. In some embodiments, R12 and R18 are independently H
or alkyl
(preferably, unsubstituted C1-C4 alkyl). Preferably, R12 and R18 are Ci_4
alkyl, such as methyl
or ethyl. In some embodiments, w is 0. It has been found experimentally that
this particular
self-immolative spacer cyclizes relatively quickly.
[00154] In some embodiments, L1 or L2 includes
CI=\ /0¨CN/R17
N4
R24
where R12, Ris, R19, K-24,
and K are as defined above.
(iii) Spacer Groups
[00155] The spacer group L3 comprises a primary or secondary amine or
a
carboxyl functional group, and either the amine of L3 forms an amide bond with
a pendant
carboxyl functional group of D or the carboxyl of L3 forms an amide bond with
a pendant
amine functional group of D. L3 can be selected from the group consisting of
substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted aryl,
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substituted or unsubstituted heteroaryl, or substituted or unsubstituted
heterocycloalkyl. In a
preferred embodiment, L3 comprises an aromatic group. More preferably, L3
comprises a
benzoic acid group, an aniline group or indole group. Non-limiting examples of
structures
that can serve as an - L3-NH¨ spacer include the following structures:
HN k- HN k
= .
0
01111
NH 1
\ Z
0
41 NH ,
0 is
\ z \ z
4 NH
0
where Z is a member selected from 0, S and NR23, and where R23 is a member
selected from
H, substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, and acyl.
[00156] Upon
cleavage of the linker of the invention containing L3, the L3
moiety remains attached to the drug, D. Accordingly, the L3 moiety is chosen
such that its
attachment to D does not significantly alter the activity of D. In another
embodiment, a
portion of the drug D itself functions as the L3 spacer. For example, in one
embodiment, the
drug, D, is a duocarmycin derivative in which a portion of the drug functions
as the L3 spacer.
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Non-limiting examples of such embodiments include those in which NH2-( L3)-D
has a
structure selected from the group consisting of:
NI-I2
CO2Me
Cl
HN
HO 1 1 N 110 0
0
where Z is 0, S or N R23, where R23 is H, substituted or unsubstituted alkyl,
substituted or
unsubstituted heteroalkyl, or acyl; and the NH2 group on each structure reacts
with (AA1), to
form -(AA1), -NH¨.
(iv) Peptide Sequence (AA1),
[00157] The
group AA1 represents a single amino acid or a plurality of amino
acids joined together by amide bonds. The amino acids may be natural amino
acids and/or
unnatural a-amino acids. They may be in the L or the D configuration. In one
embodiment, at
least three different amino acids are used. In another embodiment, only two
amino acids are
used.
[00158] The term
"amino acid" refers to naturally occurring and synthetic
amino acids, as well as amino acid analogs and amino acid mimetics that
function in a
manner similar to the naturally occurring amino acids. Naturally occurring
amino acids are
those encoded by the genetic code, as well as those amino acids that are later
modified, e.g.,
hydroxyproline, y-carboxyglutamate, citrulline, and 0-phosphoserine. Amino
acid analogs
refers to compounds that have the same basic chemical structure as a naturally
occurring
amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group,
an amino group,
and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine
methyl
sulfonium Such analogs have modified R groups (e.g., norleucine) or modified
peptide
backbones, but retain the same basic chemical structure as a naturally
occurring amino acid.
One amino acid that may be used in particular is citrulline, which is a
precursor to arginine
and is involved in the formation of urea in the liver. Amino acid mimetics
refers to chemical
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compounds that have a structure that is different from the general chemical
structure of an
amino acid, but functions in a manner similar to a naturally occurring amino
acid. The term
"unnatural amino acid" is intended to represent the "D" stereochemical form of
the twenty
naturally occurring amino acids described above. It is further understood that
the term
unnatural amino acid includes homologues of the natural amino acids, and
synthetically
modified forms of the natural amino acids. The synthetically modified forms
include, but are
not limited to, amino acids having alkylene chains shortened or lengthened by
up to two
carbon atoms, amino acids comprising optionally substituted aryl groups, and
amino acids
comprised halogenated groups, preferably halogenated alkyl and aryl groups.
When attached
to a linker or conjugate of the invention, the amino acid is in the form of an
"amino acid side
chain", where the carboxylic acid group of the amino acid has been replaced
with a keto
(C(0)) group. Thus, for example, an alanine side chain is ¨C(0) ¨CH(NH2) ¨CH3,
and so
forth.
[00159] The
peptide sequence (AA1), is functionally the amidification residue
of a single amino acid (when c=1) or a plurality of amino acids joined
together by amide
bonds. The peptide sequence (AA1), preferably is selected for enzyme-catalyzed
cleavage by
an enzyme in a location of interest in a biological system. For example, for
conjugates that
are targeted to but not internalized by a cell, a peptide is chosen that is
cleaved by a protease
that is in the extracellular matrix, e.g., a protease released by nearby dying
cells or an
amyloid-associated protease, e.g. membrane bound metalloproteases such as
alpha secretase
which cleaves APP at the cell surface, such that the peptide is cleaved
extracellularly. For
conjugates that are designed for internalization by a cell, the sequence
(AA1), preferably is
selected for cleavage by an endosomal or lysosomal protease. The number of
amino acids
within the peptide can range from 1 to 20; but more preferably there will be 1-
8 amino acids,
1-6 amino acids or 1, 2, 3 or 4 amino acids comprising (AA1),. Peptide
sequences that are
susceptible to cleavage by specific enzymes or classes of enzymes are well
known in the art.
[00160]
Preferably, (AA1), contains an amino acid sequence ("cleavage
recognition sequence") that is a cleavage site by the protease. Many protease
cleavage
sequences are known in the art. See, e.g., Matayoshi et al. Science 247: 954
(1990); Dunn et
al. Meth. Enzymol. 241: 254 (1994); Seidah et al. Meth. Enzymol. 244: 175
(1994);
Thornberry, Meth. Enzymol. 244: 615 (1994); Weber et al. Meth. Enzymol. 244:
595 (1994);
Smith et al. Meth. Enzymol. 244: 412 (1994); Bouvier et al. Meth. Enzymol.
248: 614 (1995),
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Hardy et al., in Amyloid Protein Precursor in Development, Aging, and
Alzheimer's Disease,
ed. Masters et al. pp. 190-198 (1994).
[00161] The
peptide typically includes 3-12 (or more) amino acids. The
selection of particular amino acids will depend, at least in part, on the
enzyme to be used for
cleaving the peptide, as well as, the stability of the peptide in vivo. One
example of a suitable
cleavable peptide is 13-Ala-Leu-Ala-Leu (SEQ ID NO: 1). This can be combined
with a
stabilizing group to form succiny1-13-Ala-Leu-Ala-Leu (SEQ ID NO: 2). Other
examples of
suitable cleavable peptides are provided in the references cited below.
Alternatively, linkers
comprising a single amino acid residue can be used, as disclosed in WO
2008/103693.
[00162]i i
In a preferred embodiment, the peptide sequence (AA ), s chosen
based on its ability to be cleaved by a lysosomal proteases, examples of which
include
cathepsins B, C, D, H, L and S. Preferably, the peptide sequence (AA1), is
capable of being
cleaved by cathepsin B or serine protease neurosin, in vitro. Caspase cleavage
of Tau protein.
Also, serine protease neurosin (kallikrein-6) degrades a-synuclein and co-
localizes with
pathological inclusions such as Lewy bodies and glial cytoplasmic inclusions.
[00163] A
growing amount of evidence indicates that MMPs may play an
important role in the pathogenesis of Alzheimer's disease (AD). Peptide
sequences designed
to be cleaved by matrix metalloproteases (MMP)-2 and MMP-9 have been designed
and
tested for conjugates of dextran and methotrexate; PEG (polyethylene glycol)
and
doxorubicin (Bae et al., Drugs Exp. Clin. Res. 29:15-23 (2004)); and albumin
and
doxorubicin (Kratz et al., Bioorg. Med. Chem. Lett. 11:2001-2006 (2001)).
Examples of
suitable sequences for use with MMPs include, but are not limited to, Pro-Val-
Gly-Leu-Ile-
Gly (SEQ ID NO: 3), Gly-Pro-Leu-Gly-Val (SEQ ID NO: 4), Gly-Pro-Leu-Gly-Ile-
Ala-Gly-
Gln (SEQ ID NO: 5), Pro-Leu-Gly-Leu (SEQ ID NO: 6), Gly-Pro-Leu-Gly-Met-Leu-
Ser-Gln
(SEQ ID NO: 7), and Gly-Pro-Leu-Gly-Leu-Trp-Ala-Gln (SEQ ID NO: 8). See, e.g.,
the
previously cited references as well as Kline et al. (2004) Mol. Pharmaceut.
1:9-22 and Liu et
al. (2000) Cancer Res. 60:6061-6067.
[00164] Yet
another example is type II transmembrane serine proteases. This
group of enzymes includes, for example, hepsin, testisin, and TMPRSS4. Gln-Ala-
Arg is one
substrate sequence that is useful with matriptase/MT-SP1 and Leu-Ser-Arg is
useful with
hepsin. (See, e.g., Lee et. al. (2000) J. Biol. Chem. 275:36720-36725 and
Kurachi and
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Yamamoto, Handbook of Proeolytic Enzymes Vol. 2, 2nd edition (Barrett A J,
Rawlings N D
& Woessner J F, eds) pp. 1699-1702 (2004)).
[00165]
Suitable, but non-limiting, examples of peptide sequences suitable for
use in the conjugates of the invention include Val-Cit, Cit-Cit, Val-Lys, Phe-
Lys, Lys-Lys,
Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N9-tosyl-Arg, Phe-
N9-nitro-Arg,
Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-
Val, Ala-
Len-Ala-Leu, 13-Ala-Leu-Ala-Leu (SEQ ID NO: 9), Gly-Phe-Leu-Gly (SEQ ID NO:
10), Val-
Ala, Leu-Leu-Gly-Leu (SEQ ID NO: 11), Leu-Asn-Ala, and Lys-Leu-Val. Preferred
peptides
sequences are Val-Cit and Val-Lys.
[00166] In
another embodiment, the amino acid located the closest to the drug
moiety is selected from the group consisting of: Ala, Asn, Asp, Cit, Cys, Gln,
Gln, Gly, Ile,
Len, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. In yet another
embodiment, the amino
acid located the closest to the drug moiety is selected from the group
consisting of: Ala, Asn,
Asp, Cys, Gln, Gln, Gly, Ile, Leu, Met, Phe, Pro, Ser, Thr, Tip, Tyr, and Val.
[00167] One of
skill in the art can readily evaluate an array of peptide
sequences to determine their utility in the present invention without resort
to undue
experimentation. See, for example, Zimmerman, M., et al., (1977) Analytical
Biochemistry
78:47-51; Lee, D., et al., (1999) Bioorganic and Medicinal Chemistry Letters
9:1667-72; and
Rano, T. A., et al., (1997) Chemistry and Biology 4:149-55.
[00168] A
conjugate of this invention may optionally contain two or more
linkers. These linkers may be the same or different. For example, a peptidyl
linker may be
used to connect the drug (i.e., cytoprotective agent) to the ligand (i.e.,
antibody) and a second
peptidyl linker may attach a diagnostic agent (e.g., marker) to the antibody
partner conjugate.
Other uses for additional linkers include linking analytical agents,
biomolecules, targeting
agents, and detectable labels to the antibody-partner complex.
(v) Hydrazine Linkers (H)
[00169] In
another embodiment, the conjugate of the invention comprises a
hydrazine self-immolative linker, wherein the conjugate has the structure:
X4(L4)H( L1)õ-D
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wherein D, L1, L4, p, m, and X4 are as defined above and described further
herein, and H is a
linker comprising the structure:
(R24)3c R24
R24 R24 k24
1 m 1
(....õ--............).;=:õ.õ .....,N N
"...õ,,
0 I
112
'`......
lyz 1 0.........,,.........
0 0
R24 R24
wherein n1 is an integer from 1-10; nz is 0, 1, or 2; each R24 is a member
independently
selected from the group consisting of H, substituted alkyl, unsubstituted
alkyl, substituted
heteroalkyl, and unsubstituted heteroalkyl; and I is either a bond (i.e., the
bond between the
carbon of the backbone and the adjacent nitrogen) or:
R24 R24
R24 R24
0
wherein 113 is 0 or 1, with the proviso that when 113 is 0, nz is not 0; and
114 is 1, 2, or 3.
[00170] In one embodiment, the substitution on the phenyl ring is a
para
substitution. In certain preferred embodiments, 111 is 2, 3, or 4 or 'Ii is 3.
In certain preferred
embodiments, nz is 1. In certain preferred embodiments, I is a bond (i.e., the
bond between
the carbon of the backbone and the adjacent nitrogen). In one aspect, the
hydrazine linker, H,
can form a 6-membered self immolative linker upon cleavage, for example, when
113 is 0 and
114 is 2. In another aspect, the hydrazine linker, H, can form two 5-membered
self immolative
linkers upon cleavage. In yet other aspects, H forms a 5-membered self
immolative linker, H
forms a 7-membered self immolative linker, or H forms a 5-membered self
immolative linker
and a 6-membered self immolative linker, upon cleavage. The rate of cleavage
is affected by
the size of the ring formed upon cleavage. Thus, depending upon the rate of
cleavage desired,
an appropriate size ring to be formed upon cleavage can be selected.
[00171] Another hydrazine structure, H, has the formula:
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RI24
--ezzcl R24 0 1
R24
where q is 0, 1, 2, 3, 4, 5, or 6; and each R24 is a member independently
selected from the
group consisting of H, substituted alkyl, unsubstituted alkyl, substituted
heteroallyl, and
unsubstituted heteroallyl. This hydrazine structure can also form five-, six-,
or seven-
membered rings and additional components can be added to form multiple rings.
[00172] The
preparation, cleavage chemistry and cyclization kinetics of the
various hydrazine linkers is disclosed in WO 2005/112919. As stated above,
upon the
hydrazine moiety is converted to a hydrazone when attached. This attachment
can occur, for
example, through a reaction with a ketone group on the L moiety. Therefore,
the term
"hydrazone linker" can also be used to describe the hydrazine linkers of the
current invention.
(vi) Disulfide Linkers (J)
[00173] In yet
another embodiment, the linker comprises an enzymatically
cleavable disulfide group. In one embodiment, the invention provides a
cytoprotectiye
antibody-partner compound haying a structure according to Formula (d):
wherein D, L1, L4, p, m, and X4are as defined above and described further
herein, and J is a
disulfide linker comprising a group haying the structure:
R24 R24 .1V111.r.IIMP
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wherein each R24 is a member independently selected from the group consisting
of H,
substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, and
unsubstituted heteroalkyl;
each K is a member independently selected from the group consisting of
substituted alkyl,
unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl,
substituted aryl,
unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl,
substituted
heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, NO2, NR21R22,
NR21c0R22,
0C0NR21R22, 000R21, and OR21 wherein R21 and R22 are independently selected
from the
group consisting of H, substituted alkyl, unsubstituted alkyl, substituted
heteroalkyl,
unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted
heteroaryl,
unsubstituted heteroaryl, substituted heterocycloalkyl and unsubstituted
heterocycloalkyl; i is
an integer of 0, 1, 2, 3, or 4; and d is an integer of 0, 1, 2, 3, 4, 5, or 6.
[00174] The aromatic ring of a disulfide linker can be substituted
with one or
more "K" groups. A "K" group is a substituent that replaces a hydrogen
otherwise attached to
one of the four non-substituted carbons that are part of the ring structure.
The "K" group may
be a single atom, such as a halogen, or may be a multi-atom group, such as
alkyl, heteroalkyl,
amino, nitro, hydroxy, alkoxy, haloalkyl, and cyano. Exemplary K substituents
include, but
are not limited to, F, Cl, Br, I, NO2, OH, OCH3, NHCOCH3, N(CH3)2, NHCOCF3 and
methyl. For "Ki", i is an integer of 0, 1, 2, 3, or 4. In a specific
embodiment, i is 0.
[00175] In a preferred embodiment, the linker comprises an
enzymatically
cleavable disulfide group of the following formula:
0 R24 R24
X4
S =
s K
R24 R24
wherein L4, X4, p, and R24 are as described above, and d is 0, 1, 2, 3, 4, 5,
or 6. In a particular
embodiment, d is 1 or 2.
[00176] A more specific disulfide linker is shown in the formula
below:
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0
24
R24 R24
S
d S
Preferably, d is 1 or 2 and each K is H.
[00177] Another disulfide linker is shown in the formula below:
R24 R24 R24
0
\KS S
¨ K
Preferably, d is 1 or 2 and each K is H.
[00178] In various embodiments, the disulfides are functionalized at
the ortho
position. In another specific embodiment, d is 0. In certain preferred
embodiments, R24 is
independently selected from H and CH3.
[00179] The preparation and use of disulfide linkers such as those
described
above is disclosed in WO 2005/112919.
[00180] In a specific embodiment, a linker of the invention is
selected from the
group constising of peptide-based linkers such as citrulline-valine linkers,
(dervived from
maleimidocaproyl-valinecitrulline-p-aminobenzyloxycarbonyl), and linkers
derived from
lysine residues , disulfide linkers derived from N-succinimydyl 4-(2-
pyridyldithio)-
pentanoate (SPP), and thioether linkers derived from succinimidy1-4-(N-
maleimidomethyl)cyclohexane- 1 -carboxylate (MCC), bis-maleimido-
trioxyethylene glycol
[see, Junutula et al. (2010) Clin Cancer Res; 16(19); 4769-78]; , or
maleimidocaproyl.
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[00181] For
further discussion of types of linkers and the conjugation of
therapeutic agents to antibodies, see also U.S. Pat. No. 7,087,600; U.S. Pat.
No. 6,989,452;
U.S. Pat. No. 7,129,261; US 2006/0004081; US 2006/0247295; WO 02/096910; WO
2007/051081; WO 2005/112919; WO 2007/059404; WO 2008/083312; WO 2008/103693;
Saito et al. (2003) Adv. Drug Deliv. Rev. 55:199-215; Trail et al. (2003)
Cancer Immunol.
Immunother. 52:328-337; Payne. (2003) Cancer Cell 3:207-212; Allen (2002) Nat.
Rev.
Cancer 2:750-763; Pastan and Kreitman (2002) Curr. Opin. Investig. Drugs
3:1089-1091;
Senter and Springer (2001) Adv. Drug Deliv. Rev. 53:247-264.
(IV.) Methods of Conjugation
[00182] Partner
molecules and antibodies of the invention may be conjugated
using any suitable technique known in the art. For example, the Mannich
reaction offers the
possibility of conjugating melatonin directly to solvent accessible lysine
residues. The
Mannich reaction consists of the condensation of formaldehyde (or sometimes
another
aldehyde) with ammonia, in the form of its salt, and another compound
containing an active
hydrogen. See, Vollhardt and Schore, Organic Chemistry: Structure and
Function, Fourth
Edition, W. H. Freeman & Co. 0 2002 W. H. Freeman & Co., and Sumanas, Inc.
Instead of
using ammonia, however, this reaction can also be done with primary or
secondary amines,
or even with amides. An example of this reaction is illustrated in the
condensation of
acetophenone, formaldehyde, and a secondary amine salt: C6H5COCH3 + CH20 +
R2NH=FIC1 -C6H5COCH2CH2NR2=HC1 + H20. Melatonin was previously conjugated to
bovine serum albumin using this method, and can involve cross-linking at
position 1 of the
indole ring to the free amine of lysine residues in the protein. See, Amines
and their
metabolites (1985) by Alan A. Boulton, Glen B. Baker, Judith M. Baker, page
271.
[00183] The
skilled artisan will understand that the specific method to be used
for conjugation of antibody to partner molecule will depend up on the specific
linker and
partner molecule being used. By way of example, and without limitation,
provided herein is a
description of conjugation of an antibody to a drug using Succinimidy1-4-(N-
maleimidomethyl)-cyclohexane- 1-carboxylate (SMCC, Pierce, Rockford, IL). SMCC
is
dissolved in dimethylacetamide (DMA) and added to the antibody solution to
make a final
SMCC/Ab molar ratio of 10:1. The reaction is allowed to proceed for 3 hours at
room
temperature with mixing. The SMCC-modified antibody is subsequently purified
on a GE
Healthcare HiTrap desalting column (G-25) equilibrated in 35 mM sodium citrate
with 150
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mM NaC1 and 2 mM EDTA, pH 6Ø The drug (e.g. melatonin) is dissolved in DMA,
and
added to the SMCC-antibody preparation to give a molar ratio of melatonin to
antibody of
10:1. The reaction is allowed to proceed for 4 to 20 hours at room temperature
with mixing.
The melatonin-modified antibody solution is diafiltered with 20 volumes of
phosphate-
buffered saline to remove unreacted melatonin, sterile-filtered, and stored at
4 C. Typically, a
40% to 60% yield of antibody is achieved through this process. The preparation
is usually
greater than 95% monomeric as may be assessed by gel filtration and laser
light scattering.
Typically, the drug to antibody ratio is expected to be between about 2.5 and
4.5. See, Polson
et al. (2007) Blood 110(2)616-623.
(IV.) Pharmaceutical Compositions
[00184] While
compositions of the invention may be administered alone, it may
be preferable in some embodiments to administer them as a pharmaceutical
formulation.
[00185]
Pharmaceutical compositions include an active agent and a
pharmaceutically acceptable carrier, excipient, or diluent. Pharmaceutically
acceptable
carriers, including diluents or excipients, for therapeutic use are well known
in the
pharmaceutical art, and are described herein and, for example, in Remington's
Pharmaceutical
Sciences, Mack Publishing Co. (A.R. Gennaro, ed., 18th Edition (1990)) and in
CRC
Handbook of Food, Drug, and Cosmetic Excipients, CRC Press LLC (S.C.
Smolinski, ed.
(1992)). The term "carrier" applied to pharmaceutical compositions of the
invention refers to
a diluent, excipient, or vehicle with which a compound is administered. Such
pharmaceutical
carriers can be sterile liquids, such as water and oils, including those of
petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame oil and the
like. Water or aqueous solution, saline solutions, and aqueous dextrose and
glycerol solutions
are preferably employed as carriers, particularly for injectable solutions.
Suitable
pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences"
by E.W.
Martin, 18th Edition.
[00186] For
human therapy, the pharmaceutical compositions, including each
of the active agents, will be prepared in accordance with good manufacturing
process (GMP)
standards, as set by the Food & Drug Administration (FDA). Quality assurance
(QA) and
quality control (QC) standards will include testing for purity and function
and other standard
measures.
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[00187] A
preferred delivery vehicle is any chemical entity that ensures
delivery of an inhibitor to the target site (e.g., brain, or more
specifically, an amyloid deposit
or neurofibrillary tangle) in a selective manner, achieves sufficient
concentration of active
inhibitor at the target site, and is preferably bioavailable in the brain.
[00188] In
certain embodiments, it is preferred that the compositions of the
invention be able to cross the blood brain barrier (BBB), in order to
facilitate treatment of
neuropathogenic diseases and disorders. The usefulness of IV Ig treatment in
epilepsy was
assessed by Engelen B G et al. (J. Neurol Neurosurg Psychiatry 1994 November
57 Supp 21-
5). The conclusion of these authors from the study on cerebrospinal fluid IgG
concentrations
before and after IV Ig treatment in patients with epilepsy was that the main
component of IV
Ig preparation crosses the blood CSF barrier and significantly increases CSF
IgG
concentration, and may reach the brain and act centrally. The presence of IgG
in the CNS was
demonstrated by immunocytochemistry and showed a close anatomical relationship
between
the distribution of this protein and the blood-brain barrier. IgG was
immunolocalized in the
normal rat brain by using monoclonal and polyclonal antibodies to IgG and its
subclasses.
Further, the passage of intravenous immunogloubulin and interactions with the
CNS was
summarized in a review by Wurster et al. (J. Neurol Neurosurg Psychiatry 1994
November
57 Supp 21-5).
[00189] In
certain embodiments, the ADC of the invention penetrate into the
brain cells, through the blood brain barrier (BBB), by using methods or
carriers, which details
are provided below. For example, and without limitation, preferred compounds
that may be
added to formulations to enhance the solubility of the ADC of the present
invention are
cyclodextrin derivatives, preferably hydroxypropyl-gamma-cyclodextrin. Drug
delivery
vehicles containing a cyclodextrin derivative for delivery of peptides to the
central nervous
system are described in Bodor, N., et al. (1992) Science 257:1698-1700.
[00190]
Accordingly, use of an ADC of the invention in combination with a
cyclodextrin derivative may result in greater inhibition of 13 amyloid
neurotoxicity than use of
the ADC alone. Chemical modifications of cyclodextrins are known in the art
(Hanessian, S.,
et al. (1995) J. Org. Chem. 60:4786-4797). In addition to use as an additive
in an ADC-
containing composition (including pharmaceutical composition) of the
invention, cyclodextin
derivatives may also be useful as modifying groups and, accordingly, may also
be covalently
coupled to antibodies comprised in ADC of the invention.
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[00191] Low et
al., U.S. Pat. No. 5,108,921, reviews available methods for
transmembrane delivery of molecules such as proteins and nucleic acids by the
mechanism of
receptor mediated endocytotic activity. These receptor systems include those
recognizing
galactose, mannose, mannose-6-phosphate, transferrin, asialoglycoprotein,
transcobalamin
(vitamin B<sub>12</sub>), a-2 macroglobulins, insulin and other peptide growth
factors such
epidermal growth factor (EGF). Low et al. also teaches that nutrient
receptors, such as
receptors for biotin and folate, can be advantageously used to enhance
transport across the
cell membrane due to the location and multiplicity of biotin and folate
receptors on the
membrane surfaces of most cells, and the associated receptor mediated
transmembrane
transport processes. Thus, a complex formed between a compound to be delivered
into the
cytoplasm and a ligand, such as biotin or folate, is contacted with a cell
membrane bearing
biotin or folate receptors to initiate the receptor mediated trans-membrane
transport
mechanism and thereby permit entry of the desired compound into the cell.
[00192] A biotin
ligated can be attached to a DM molecule, for example, by
incorporating commercially available biotinylated deoxyuucleotide
triphosphates, e.g., biotin-
14-MTP or biotin-14dCTP from Invitrogen Life Technologies, Carlsbad, Calif.,
using
terminal deoxynucleotidyl transferase (Karger, B. D., 1989). Biotin-14dATP is
a MTP analog
with biotn attached at the 6-position of the purine base by a 14-atom linker
and biotin-14-
dCTP is a dCTP analog with biotin attached at the N<sup>4-position</sup> of the
pyrmidine base
also by a 14-atom liker.
[00193] In one
embodiment, the ADC of the invention can be delivered by
liposomes, as discussed, supra.
[00194] In
another approach for enhancing transport across the BBB, and ADC
of the invention is conjugated to a second peptide or protein thereby forming
a chimeric
protein, wherein the second peptide or protein undergoes absorptive-mediated
or receptor-
mediated transcytosis through the BBB. Accordingly, by coupling the ADC to
this second
peptide or protein, the chimeric protein is transported across the BBB. The
second peptide or
protein can be a ligand for a brain capillary endothelial cell receptor
ligand. For example, a
preferred ligand is a monoclonal antibody that specifically binds to the
transferrin receptor on
brain capillary endothelial cells (see e.g., U.S. Pat. Nos. 5,182,107 and
5,154,924 and PCT
Publications WO 93/10819 and WO 95/02421, all by Friden et al.). Other,
suitable peptides
or proteins that can mediate transport across the BBB include histones (see
e.g., U.S. Pat. No.
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4,902,505 by Pardridge and Schimmel) and ligands such as biotin, folete,
niacin, pantothenic
acid, riboflavin, thiamin, pryridoxal and ascorbic acid (see e.g., U.S. Pat.
Nos. 5,416,016 and
5,108,921, both by Heinstein). Additionally, the glucose transporter GLUT-1
has been
reported to transport glycopeptides (L-serinyl-fl-D-glucoside analogues of
[Met5]enkephalin)
across the BBB (Polt, R. et al. (1994) Proc. Natl Acad. Sci. USA 91:7114-
1778).
Accordingly, an ADC described herein can be coupled to such a glycopeptide to
target the
ADC to the GLUT-1 glucose transporter. For example, an ADC comprising an
antibody
which is modified at its amino terminus with the modifying group Aic (3-(0-
aminoethyl-iso)-
cholyl, a derivative of cholic acid having a free amino group) can be coupled
to a
glycopeptide through the amino group of Aic by standard methods. Chimeric
proteins can be
formed by recombinant DNA methods (e.g., by formation of a chimeric gene
encoding a
fusion protein) or by chemical crosslinking of the antibody of the ADC to the
second peptide
or protein to form a chimeric protein. Numerous chemical crossing agents are
known (e.g.,
commercially available from Pierce, Rockford Ill.). A crosslinking agent can
be chosen
which allows for high yield coupling of the antibody to the second peptide or
protein and for
subsequent cleavage of the linker to release bioactive ADC. For example, a
biotin-avidin-
based linker system may be used.
[00195] In yet
another embodiment for enhancing transport across the BBB, the
ADC is encapsulated in a carrier vector, which mediates transport across the
BBB. For
example, the ADC can be encapsulated in a liposome, such as a positively
charged
unilamellar liposome (see e.g., PCT Publications WO 88/07851 and WO 88/07852,
both by
Faden) or in polymeric microspheres (see e.g., U.S. Pat. No. 5,413,797 by Khan
et al., U.S.
Pat. No. 5,271,961 by Mathiowitz et al. and 5,019,400 by Gombotz et al.).
Moreover, the
carrier vector can be modified to target it for transport across the BBB. For
example, the
carrier vector (e.g., liposome) can be covalently modified with a molecule
which is actively
transported across the BBB or with a ligand for brain endothelial cell
receptors, such as a
monoclonal antibody that specifically binds to transferrin receptors (see
e.g., PCT
Publications WO 91/04014 by Collins et al. and WO 94/02178 by Greig et al.).
[00196] An ADC
of the invention can be formulated into a pharmaceutical
composition wherein the ADC is the only active compound or, alternatively, the
pharmaceutical composition can contain additional active compounds. For
example, two or
more active agents may be used in combination (e.g. two or more ADC, or an ADC
in
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combination with another active agent). Moreover, an ADC of the invention can
be combined
with one or more other agents that have anti-amyloidogenic and/or anti-
fibrillogenic
properties. For example, an ADC can be combined with the non-specific
cholinesterase
inhibitor tacrine (COGNEXTM, Parke-Davis).
(VI.) Methods of Treatment
(A) Methods for Treating Proteinopathies
[00197] As
described herein, it is presently discovered that antibodies specific
for amyloid polypeptides (e.g., A13) and amyloid-associated polypeptides
(e.g., tau or APP)
can be used to target cytoprotective agents such as antioxidants, to a site of
damage in
proteinopathies (e.g., an amyloid deposit). While not intending to be bound by
theory or a
particular mechanism of action, the ADC can therefore provide the dual benefit
of the
protein-clearing action of the antibody, coupled to specific targeting of a
cytoprotective agent
to a site in need of such cytoprotection. Thus, the compositions of the
invention are useful
both for clearing amyloid deposits and for providing cytoprotective (e.g.,
neuroprotective)
effects to the targeted cell. Moreover, in a specific embodiment, the
cytoprotective agent is
melatonin, which not only provides beneficial antioxidant effects, but can
also interact with
the high affinity copper binding site in A13 and inhibit fibril formation.
[00198] In
certain aspects, the invention thus provides methods for inhibiting
accumulation of amyloid polypeptides or amyloid-associated polypeptides in the
brain of a
patient suffering from a proteinopathy. In certain embodiments, the method
comprises
contacting in vivo soluble amyloid polypeptides, amyloid-associated
polypeptides, tau, or
other polypeptide involved in a proteinopathy (i.e., target polypeptide) in
said patient with an
ADC composition of the invention. The site of contact of the ADC and the
target polypeptide
will vary depending on the route of delivery and the specific proteinopathy
being treated. In
one embodiment, the site of contact is in the cerebrospinal fluid of the
patient. In other
embodiments, such as, e.g., for treating a peripheral amyloidosis (e.g., type
II diabetes or
serum amyloidosis), the contact site will be a peripheral site (non CNS), such
as, but not
limited to, in the blood, liver, spleen, kidney, adrenal, heart, or pancreas
or other organ.
[00199] In
another embodiment, the invention provides a method for treating or
delaying onset of a proteinopathy, comprising administering to a subject in
need thereof an
effective amount of an ADC composition of the invention, to inhibit the
formation of fibrils
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or the formation of amyloid or amyloid-like deposits associated with
amyloidosis-related
diseases, or to inhibit the formation of neurofibrillary tangles associated
with tauopathies.
[00200] In
another embodiment, the invention provides a method for promoting
clearance of aggregates from the brain of a subject, comprising administering
to the subject in
need thereof an effective amount of an ADC composition of the invention,
wherein said
polypeptide is tau, under conditions and in an amount effective to promote
clearance of
neurofibrillary tangles from the brain of the subject.
[00201]
Proteinopathies that can be treated according to the present invention
include, but are not limited to : (1) neuropathic conditions associated with
A13 amyloidosis,
such as age related macular degeneration (A13), glaucoma, traumatic brain
injury, cerebral
amyloid angiopathy (CAA), hereditary cerebral hemorrhage with amyloidosis
Dutch type
(HCHWA-D) AD, early onset familial AD (EOFAD), and Down Syndrome; as well as
(with
the amyloidogenic protein involved in the disease or disorder is shown in
parentheses):
Parkinson's disease (PD) (a-synuclein), Huntington's disease (Huntingtin
protein),
amyotrophic lateral sclerosis (superoxide dismutase), Pick's complex (Tau),
familial
frontotemporal dementias (Tau) and prion disease (pathogenic prion, PrPSc);
and (2) non-
neuropathic amyloid-associated diseases and disorders, such as, but not
limited to, diabetes
mellitus type 2 (TAPP (Amylin)) (the amyloidogenic protein involved in the
disease or
disorder is shown in parentheses), medullary carcinoma of the thyroid
(calcitonin), isolated
atrial amyloidosis (atrial natriuretic factor (AANF)), atherosclerosis
(apolipoprotein AT
(AApoA1)), rheumatoid arthritis (serum amyloid A (AA)), aortic medial amyloid
(medin
(AMed)), familial amyloid polyneuropathy (transthyretin (ATTR)), hereditary
non-
neuropathic systemic amyloidosis (lysozyme (ALys)), dialysis related
amyloidosis (beta 2
microglobulin (A132M), Finnish amyloidosis (Gelsolin (AGel)), Lattice corneal
dystrophy
(keratoepithelin (AKer)), cerebral amyloid angiopathy (beta amyloid (A13)),
cerebral amyloid
angiopathy (Icelandic type) (cystatin (ACys)), systemic AL amyloidosis
(Immunoglobulin
light chain AL (AL)), and sporadic inclusion body myositis (amyloid precursor
protein, beta
amyloid, presenilinl, sequestosome 1 (p62), TAR DNA binding protein-43 (TDP-
43),
ubiquitinated-proteins, apolipoprotein E, alpha-synuclein and phosphorylated
tau).
[00202] In
certain embodiments, once delivered into the brain an ADC will
transfer into the extracellular space, interstitial fluid and cerebrospinal
fluid. The specific
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antibodies and/or cytoprotective agents (e.g., melatonin) of the ADC then form
a soluble
complex with the target polypeptide (e.g., A13 peptide, tau, prion PrPSc).
These soluble
complexes reduce, in one embodiment, the deposition of A13 peptides into
amyloid plaques
and attenuate A13 peptide-induced neurotoxicity by clearing A13 peptides from
the central
nervous system through drainage of the extracellular space, interstitial fluid
and cerebrospinal
fluid into the general blood circulation where they will be eliminated by
protease digestion.
The cytoprotective agent in the conjugate provides (e.g., melatonin) provides
additional
protective effects (e.g., antioxidant effects). Accordingly, the accumulation
of newly secreted
soluble A13 peptides responsible for amyloid deposition and A13 -induced
neurotoxicity is
inhibited and cytoprotection is conferred. ADCs can also bind directly to
insoluble plaque
causing clearance through Fc-mediated phagocytosis by microglia. In some cases
ADCs may
be internalized and bind intracellular amyloid.
[00203]
Furthermore, clearance of amyloid- 13 peptides in accordance with the
present invention is expected to reduce the inflammatory process observed in
Alzheimer's
disease and other amyloidogenic diseases or disorders by inhibiting, for
example, amyloid 13-
induced complement activation and cytokine release, and blocking also the
interaction of A13
with cell surface receptors such as the RAGE receptor.
[00204] In
another embodiment, once an ADC of the invention binds to the
amyloid 13 peptide the ADC can elicit a cellular immune response (e.g., via
activation of the
Fc receptor). Fc receptor can distinguish between an antibody, which is bound
to an antigen,
and a free antibody. The result will be that the Fc receptors will enable
accessory cells, which
are usually not capable of identifying target antigens to target and engulf
A13 thereby
eliminating the requirement for a stoichiometric relationship between antibody
and antigen.
As a consequence, less ADC will be required to penetrate the BBB for
elimination of
deposited amyloid 13 peptides.
[00205] In
another embodiment, the interaction of amyloid 13 with APOE4 gene
product will be reduced following administration of an ADC of the invention.
[00206] In
another embodiment, the pharmaceutical composition and the
antibodies of the present invention will delay the onset and inhibit or
suppress the progression
of a proteinopathy by having a peripheral effect. The clearance or the removal
of amyloid
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beta from the periphery will change the equilibrium of the amyloid beta in the
blood and as a
result in the brain. Recent studies have shown that amyloid beta is
transported from the
cerebrospinal fluid to the plasma with an elimination half-life from brain of
about half an
hour. Thus, the ADC can affect the amyloid beta level in the plasma, cause
accumulation of
central amyloid beta in the plasma and as a result reduce the amyloid 0
deposition in the
brain.
(B) Proteinopathy Assays
[00207] A number of biological assays are available to evaluate and to
optimize
the ADC compositions of the invention in vitro and in vivo.
[00208] For example, to assess the cytoprotective effect of ADC of the
invention in vitro, cells (e.g., PC12 cells ATCC (Catalog # CRL-1721)) exposed
to A13 (23-
35) or the superoxide dismutase (SOD) inhibitor diethyldithiocarbamic acid
(DDTC), which
are oxidotoxins, may be treated with an ADC of the invention (or, in parallel,
with melatonin
or antibody alone, or with media alone) and the degree of lipid peroxidation
can be measured.
Under these experimental conditions, the degree of lipid peroxidation can be
estimated by
measuring the formation of malondialdehyde acid (MDA) in cell lysates as
described. See,
Omar, R. A., et al. (1987) Cancer Res 47:3473-3476.
[00209] Binding affinity of the ADC may be measured according to any
suitable technique known in the art. The following methods are described as a
non-limiting
example. Affinity measurements of an ADC may be carried out using BiaCore
kinetic
analysis (Surface Plasmon Resonance (SPR)). For example, the ability of an ADC
to
immobilize high levels (high density) or low density of soluble A131-40 can be
measured
using a Biacore T100 using CMS sensor chips (Cat: BR-1005-30). For measuring
high
density A131-40 binding, soluble and aggregated A131-40 (Bachem H-1194 and H-
5568,
respectively) is immobilized onto a sensor chip coated with carboxymethylated
dextran
(Biacore CMS). A BSA coated flow cell may be used as a blank. Amine coupling
may be
performed as follows: activation is carried out through the injection of a 1:
1 EDC:NHS
mixture for 7 min then coupling performed by injecting ligand at 20 1.ig/mL
diluted in 100
mM sodium acetate (PH 3.8) at 10 pL/min for 7 minutes. The A13 peptides are
immobilized
until saturation. Similarly BSA (Sigma A 7030) at 30 1.ig/mL is injected at 10
pL/min for 7
minutes. The remaining activated carboxyl groups were blocked with 1 M
ethanolamine (pH
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8.5). The ADC are diluted in running buffer HBS-N in concentrations ranging
from 1000 nM
(150 [ig/mL ) to 31.25 nM and then injected at a flow rate of 20 [iL/min for 3
min. Buffer is
allowed to flow over the surface for 5 min for dissociation data. Regeneration
of the flow
cells is regenerated by a single 30 s pulse with 100mM HC1 at 10 [iL/min. The
kinetics of
binding/dissociation is analyzed according to the 1: 1 interaction model using
BIAcore T100
evaluation software package 2Ø
[00210] Low
density immobilization of soluble and aggregated A131-40 may be
assayed as follows: soluble A131-40 is immobilized as described above with the
following
modifications. A131-40 is diluted to a final concentration of 0.25 [ig/mL and
coupled for 50 s
at 10 [iL/min. Flow cell I is treated to the coupling reaction but in the
absence of peptide.
Aggregated A131-40 is treated in the same manner with flow cells 3 used as the
blank control
and peptide coupled to flow cell 4. A final response level is determined.
[00211] The SPR
conditions for affinity determination are set as follows: ADC
binding to the soluble peptide A131-40 are treated as follows: the mAbs are
diluted in running
buffer HBS-EP (GE Healthcare cat: BR-100I-88) plus 1 mg/ml of carboxymethyl
dextran
(GE Healthcare BR -1006-91) at concentrations ranging from 80 nM (12 [ig/mL)
to 1.25 nM
and then injected at a flow rate of 60 [iL/min for 160 sec. Buffer is allowed
to flow over the
surface for 10 min for dissociation data. Regeneration of the flow cells is
carried out by a
single 30 s pulse with 100mM HC1 at 10 L/min. The kinetics of
binding/dissociation can be
analyzed according to the 1:1 interaction model using BIAcore T100 evaluation
software
package 2Ø ADC binding to the aggregated peptide A131-40 may be treated as
follow:
[00212] The ADC
are diluted in running buffer HBS-EP plus [ig/m1 of
carboxymethyl dextran at concentrations ranging from 160 nM (24 [ig/mL ) to
1.25 nM and
then injected at a flow rate of 60 [iL/min for 100 sec. Buffer is allowed to
flow over the
surface for 15 min for dissociation data. Regeneration of the flow cells is
regenerated by a
single 30 s pulse with 150mM HC1 at 10 [iL/min. The kinetics of
binding/dissociation is
analyzed according to the 1:1 interaction model using BIAcore T100 evaluation
software
package 2Ø
[00213] Other
methods for assaying activity of ADC of the invention are
described, e.g., in Pappolla et al., supra, and Cheng et al., supra.
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[00214] In
certain embodiments, it may be desirable to determine the
immunogenicity of an ADC of the invention. Preferably, the ADC have low
immunogenicity.
Assays for determining whether an ADC is immunogenic are described, e.g., in
Van Walle I,
et al. (2007) Expert Opin Biol Ther. ;7:405-18. Furthermore, commercial
services for assaying
immunogenicity are available, e.g., from Lonza (Basel, Switzerland).
[00215] For
characterizing the properties of an ADC of the invention in vivo,
deposition of amyloid 13 (1-40) and amyloid 13 (1-42) or other amyloidogenic
protein may be
determined in animal models following treatment with an ADC composition of the
invention.
For such assays, suitable mammalian (e.g., murine) models may be used, such
as, e.g.,
Tg2576 mice genetically engineered to express amyloid precursor proteins (APP)
Hsiao, K,
et al. (1996) Science 274:99-102; or APP/PS1 Holcomb L., et al. (1998) Nature.
Med, 4: 97-
100; mice expressing mutant (P301L) tau protein Lewis et al. (2000) Nature
Genetics 25:402-
405; PD models, see Harvey BK, et al. Acta Neurochir SuppL 2008;101:89-92; for
HD
transgenic mice see P. Hemachandra et al. (1998) Genetics and Molecular Nature
Genetics
20:198-202; ALS mice are described, e.g., at
www.researchals.org/.../p4ljaxsod1manual2009120229aPcx.pdf In such models,
mice,
e.g., transgenic mice, that are predisposed to develop a proteinopathy, such
as AD, PD, HD,
etc., are injected with an ADC of the invention, or a control (e.g., melatonin
alone, antibody
alone, or PBS). Following the treatment course, which will depend upon the
specific animal
model, symptoms of the proteinopathy, e.g., appearance of amyloid deposits are
assessed,
using, e.g., immunohistochemistry or other imaging techniques known in the
art. ELISA
assays can also be used to quantify levels of amyloid 13 e.g., in brain
homogenate samples.
See, Arendash et al. (2001) DNA and Cell Biology 20(11)737-744. Other suitable
animal
models of proteinopathies are known in the art, and are also included. The
above-described
animal models are provided as examples and are not limiting.
[00216]
Preferably, an ADC composition of the invention reduces amyloid 13
deposition by at least 10%, at least 15%, at least 20%, at least 25%, at least
30%, at least
35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at
least 65%, at least
75%, or more.
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(C) In Vivo Imaging
[00217] In certain embodiments ADC of the invention are useful for in
vivo
imaging. In particular, an ADC comprising a labeled partner molecule (e.g.,
labeled
antioxidant molecule such as melatonin labeled with, e.g., a radiolabel, a
fluorescent label
(e.g., GFP), or an enzyme that catalyzes a detectable modification to a
substrate (e.g.,
horseradish peroxidase)) may be administered to a subject for diagnostic
purposes. For
example, it may be desirable to determine whether a subject (e.g., a subject
with a suspected
AD or PD diagnosis) has detectable amyloid or amyloid-like deposits in the
brain (or other
site, e.g., pancreas, kidneys, liver, etc.). An ADC comprising an antibody
targeted to a
polypeptide found in the amyloid deposit (e.g., Afl or other amyloid-
associated protein) and
containing a detectable marker may be administered to the subject, and imaging
can be
carried out according to suitable methods known in the art.
[00218] Imaging in human brains using positron emission tomography
(PET) is
described in detail, for example, in Wagner et al. (1983) Science
221(4617):1264-1266.
Wagner describes intravenous injection of 18F or 11C labeled compounds, which
are then
detected in vivo by PET. Such radiolabels and other suitable markers are
contemplated for
use herein. In animal models, ADC of the invention may be used for diagnostic
imaging by
MRI or PET or other suitable method of detection known in the art.
[00219] While it is possible that the antibody in the ADC can be
directly
labeled with a marker, in certain embodiments, it is preferred that the
partner molecule is
labeled with the marker. In certain embodiments, the partner molecule is a
small molecule
that can be more easily labeled compared to the antibody.
[00220] In a particularly preferred embodiment, the cytoprotective
partner
molecule (e.g., melatonin or indole-3-propionic acid, etc.) is directly
synthesized with a
marker (e.g., radiolabel, such as 18F, 11C or 14C). Direct synthesis of the
cytoprotective agent
to contain a detectable marker advantageously avoids the need for an
additional conjugation
step when preparing the ADC of the invention. Of course, it is to be
understood that certain
markers are preferably conjugated to the partner molecule after synthesis of
the partner
molecule.
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(VII.) Administration and Dosage
[00221] ADC-
containing compositions of the invention can be subcutaneously,
intravenously, intradermally, intramuscularly, intaperitoneally,
intracerebrally, intranasally,
orally, transdermally, buccally, intra-arterially, intracranially, or
intracephalically. A
particularly preferred route of administration of an ADC is intravenous.
[00222] An
individual in need thereof is, for example, a human or other
mammal that would benefit by the administration of an ADC described herein,
such as a
human or other mammal suffering from or at risk of developing a proteinopathy
as described
herein (e.g., AD, PD, HD, ALS, prion disease, etc.), as determined, e.g., by
the individual's
physician.
[00223]
According to this disclosure, an ADC-containing composition
(including pharmaceutical compositions) of this disclosure can be introduced
parenterally,
transmucosally, e.g., orally (per os), nasally, or rectally, or transdermally.
Parental routes
include intravenous, intra-arteriole, intra-muscular, intradermal,
subcutaneous,
intraperitoneal, intraventricular, and intracranial administration. Specific
organs may be
targeted, e.g., brain, by direct administration to the targeted organ.
[00224] For oral
administration (e.g., buccal), the pharmaceutical compositions
may take the form of tablets or capsules prepared by conventional means with
pharmaceutically acceptable excipients such as binding agents (e.g.,
pregelatinized maize
starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,
lactose,
microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g.,
magnesium
stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch
glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by
methods well
known in the art. Liquid preparations for oral administration may take the
form of, for
example, solutions, syrups or suspensions, or they may be presented as a dry
product for
constitution with water or other suitable vehicle before use. Such liquid
preparations may be
prepared by conventional means with pharmaceutically acceptable additives such
as
suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated
edible fats);
emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g.,
almond oil, oily
esters, ethyl alcohol or fractionated vegetable oils); and preservatives
(e.g., methyl or propyl-
p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer
salts, flavoring,
coloring and sweetening agents as appropriate.
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[00225]
Preparations for oral administration may be suitably formulated to give
controlled release of the active compound. For buccal administration the
compositions may
take the form of tablets or lozenges formulated in conventional manner. For
administration by
inhalation, the chaperones for use according to the present invention are
conveniently
delivered in the form of an aerosol spray presentation from pressurized packs
or a nebulizer,
with the use of a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case
of a pressurized
aerosol the dosage unit may be determined by providing a valve to deliver a
metered amount.
Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator
may be formulated
containing a powder mix of the compound and a suitable powder base such as
lactose or
starch.
[00226] The
pharmaceutical compositions may be added to a retained
physiological fluid such as blood or synovial fluid.
[00227] In
another embodiment, the active ingredient can be delivered in a
vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990);
Treat et al., in
Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler
(eds.), Liss: New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-
327; see
generally ibid.).
[00228] ADC-
containing compositions may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous infusion.
Formulations for
injection may be presented in unit dosage form, e.g., in ampoules or in multi-
dose containers,
with an added preservative. The compositions may take such forms as
suspensions, solutions
or emulsions in oily or aqueous vehicles, and may contain formulatory agents
such as
suspending, stabilizing and/or dispersing agents. Alternatively, the active
ingredient may be
in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-
free water,
before use.
[00229] In
addition to the formulations described previously, ADC-containing
compounds and compositions may also be formulated as a depot preparation. Such
long
acting formulations may be administered by implantation (for example
subcutaneously or
intramuscularly) or by intramuscular injection. Thus, for example, the
compounds and
compositions may be formulated with suitable polymeric or hydrophobic
materials (for
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example, as an emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble
derivatives, for example, as a sparingly soluble salt. In yet another
embodiment, the
therapeutic compound can be delivered in a controlled release system. For
example, an ADC-
containing composition may be administered using intravenous infusion with a
continuous
pump, in a polymer matrix such as poly-lactic/glutamic acid (PLGA), a pellet
containing a
mixture of cholesterol and the active ingredient (SilasticR.TM.; Dow Corning,
Midland,
Mich.; see U.S. Pat. No. 5,554,601) implanted subcutaneously, an implantable
osmotic pump,
a transdermal patch, liposomes, or other modes of administration.
[00230]
Administration of ADC-containing compositions may be once a day,
twice a day, or more often, but frequency may be decreased during a
maintenance phase of
the disease or disorder, e.g., once every second or third day instead of every
day or twice a
day. The dose and the administration frequency will depend on the clinical
signs, which
confirm maintenance of the remission phase, with the reduction or absence of
at least one or
more preferably more than one clinical signs of the acute phase known to the
person skilled
in the art. More generally, dose and frequency will depend in part on
recession of
pathological signs and clinical and subclinical symptoms of a disease
condition or disorder
contemplated for treatment with the present compounds.
[00231] For in
vivo prevention of cytotoxic effects, a preferred dosage of
melatonin, when administered by itself, is between about 1 p..g and about 100
g of melatonin.
Desirable serum concentrations of melatonin are in the range of about 50 1.1.M
to about 100
M. The actual preferred amount of melatonin to be administered according to
the present
invention will vary according to the particular form of melatonin (for
example, melatonin or
an analog thereof), the particular composition formulated, and the mode of
administration.
Specifically, ADC of the invention advantageously achieve high localized
concentration of
melatonin or other cytoprotective agent. Thus, in preferred embodiments, the
dosage of
melatonin is between about 0.01 ng and 1 pig. A preferred concentration of
antibody in the
ADC conjugate ranges from about 0.05 mg/kg to 10 mg/kg.
[00232] Many
factors that may modify the action of a composition of the
invention can be taken into account by those skilled in the art; e.g., body
weight, sex, diet,
time of administration, route of administration, rate of excretion, condition
of the subject,
drug combinations, and reaction sensitivities and severities. Administration
can be carried out
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continuously or periodically within the maximum tolerated dose. Optimal
administration
rates for a given set of conditions can be ascertained by those skilled in the
art using
conventional dosage administration tests.
* * *
[00233] The
present invention is also described and demonstrated by way of the
following examples. However, the use of these and other examples anywhere in
the
specification is illustrative only and in no way limits the scope and meaning
of the invention
or of any exemplified term. Likewise, the invention is not limited to any
particular preferred
embodiments described here. Indeed, many modifications and variations of the
invention may
be apparent to those skilled in the art upon reading this specification, and
such variations can
be made without departing from the invention in spirit or in scope. The
invention is therefore
to be limited only by the terms of the appended claims along with the full
scope of
equivalents to which those claims are entitled.
EXAMPLES
[00234] Reagents
and solvents used below can be obtained by commercial
sources such as Aldrich Chemical Co. (Milwaukee, Wis., USA). Mass spectrometry
results
are reported as the ratio of mass over charge for the M+H ion containing the
most common
atomic isotopes.
Example 1: (2R)-2-amino-3-41-(6-42-(5-methoxy-1H-indo1-3-ypethyl)amino)-6-
oxohexyl)-2,5-dioxopyrrolidin-3-ypthio)propanoic acid
0
H2N 0 NH
HO2C).\ScliN I
H .
0
OMe
Step 1. Preparation of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-y1)-N-(2-(5-
methoxy-1H-indol-
3-yl)ethyl)hexanamide)
0-
0 H
=
\
0 NH
0
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[00235] To a
solution of 2,5-dioxopyrrolidin-1 -y16-(2,5-dioxo-2,5-dihydro-1H-
pyrrol-1-yl)hexanoate (247 mg, 0.81 mmol) in dichloromethane (10 mL) was added
2-(5-
methoxy-1H-indo1-3-yl)ethanamine (152 mg, 0.81 mmol) and the mixture was
stirred at rt for
minutes. The succinimide precipitated as a white solid and LCMS analysis
indicated an
89% yield of the desired product and an 11% yield of the side product formed
by addition of
the amine to the double bond. The reaction mixture was filtered and
concentrated in vacuo to
afford 6-(2,5 -
dioxo-2,5 -dihydro-1H-pyrrol-1 -y1)-N-(2 -(5-methoxy-1H-indo1-3 -
yl)ethyl)hexanamide) (300 mg, isolated yield 86%, purity 88%) as a yellow oil.
MS 384.2
(M + H+).
[00236] Step 2.
Preparation of (2R)-2-amino-3-(0-(642-(5-methoxy-1H-
indo1-3-yOethyl)amino)-6-oxohexyl)-2,5-dioxopyrrolidin-3-yOthio)propanoic acid
0
HO2C1 'S-cfiH
N I
*
0
OMe
[00237] 6-(2,5 -dioxo-2,5 -dihydro-1H-pyrrol-1-y1)-N-(2 -(5 -methoxy-
1H-indol-
3-yl)ethyl)hexanamide) (307 mg, 0.801 mmol) was dissolved in N,N-
dimethylformamide
(dry) (10 mL). L-cystein (388 mg, 3.2 mmol) was added and the mixture was
stirred at rt
overnight. The mixture was concentrated in vacuo to afford the crude product
as a white
solid. Purification by flash column chromatography (5-100% MeCN in H20) and
subsequent
lyophilization afforded (2R)-2-amino-3 -((1 -(6-((2-(5 -methoxy-1H-indo1-3 -
yl)ethyl)amino)-6-
oxohexyl)-2,5-dioxopyrrolidin-3-yl)thio)propanoic acid as a white solid (100
mg, isolated
yield 24.75%). MS 505.2 (M + H+).
Example 2: (2R)-2-
amino-3-01-(1-(5-methoxy-1H-indo1-3-y1)-4,20-dioxo-7,10,13,16-
tetraoxa-3,19-diazadocosan-22-y1)-2,5-dioxopyrrolidin-3-yl)thio)propanoic acid
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0
-0 0 f.....\-OH
110 1 H H
NH2
I
HN 0 0 0
Step 1. Preparation of 1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-Apropanamido)-N-
(2-(5-
methoxy-lH-indol-3-yOethyl)-3,6,9,12-tetraoxapentadecan-15-amide
0 0--
H H
clfl N -e-Oc)Or N
1 it
0 0 0 I
NH
[00238] 2,5 -dioxopyrro lidin-1 -yl 1 -
(2,5 -dioxo-2,5-dihydro-1H-pyrrol-1 -y1)-3 -oxo-
7,10,13,16-tetraoxa-4-azanonadecan-19-oate (200 mg, 0.389 mmol) and 2-(5-
methoxy-1H-
indo1-3-yl)ethanamine (70.4 mg, 0.370 mmol) were dissolved in N,N-
dimethylformamide (2
mL) and stirred at rt for 30 min. The reaction was then filtered and
concentrated in vacuo to
afford 1-
(3 -(2,5 -dioxo-2,5 -dihydro-1H-pyrrol-1 -yl)propanamido)-N-(2-(5-methoxy-1H-
indo1-3-yl)ethyl)-3 ,6,9,12 -tetraoxapentadecan-15-amide as a yellow oil (210
mg, 92% yield).
MS 589.2 (M + H+).
[00239] Step 2. Preparation of (2R)-2-amino-34(1-(1-(5-methoxy-1H-indo1-3-y1)-
4,20-
dioxo-7,10,13,16-tetraoxa-3,19-diazadocosan-22-y1)-2,5-dioxopyrrolidin-3-
Athio)propanoic
acid
0
¨0 0 /.......\---OH
NH2
I
HN 0 0 0
[00240] 1 -(3 -(2,5 -dioxo-2,5 -dihydro-1H-pyrrol-1-
yl)propanamido)-N-(2-(5-
methoxy-1H-indo1-3 -yl)ethyl)-3,6,9,12 -tetraoxapentadec an-15 -amide (229 mg,
0.389 mmol)
was dissolved in N,N-dimethylformamide (dry) (5 mL) and combined with (R)-2-
amino-3-
mercaptopropanoic acid (189 mg, 1.556 mmol) and the mixture was stirred at rt
overnight.
The mixture was then concentrated in vacuo and purified by reversed phase
flash
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chromatography (5-100% MeCN in H20) to afford (2R)-2-amino-3-((1-(1-(5-methoxy-
1H-
indo1-3-y1)-4,20-dioxo-7,10,13,16-tetraoxa-3,19-diazadocosan-22-y1)-2,5-
dioxopyrrolidin-3-
yl)thio)propanoic acid (59 mg, 21.37 % yield) as a white solid after
lyophilization. MS 710.2
(M + H+).
Example 3: p-Amyloid aggregation, Thioflavin T, fluorescence Assay
[00241] In this
Example, the activity of the melatonin-cysteine conjugates that
were prepared in Examples 1 and 2 were tested to determine their ability to
inhibit
fibrillogenis to the activity of melatonin in the absence and presence of
ApoE4.
[00242] In a
NUNC PP 96-well plate 100 L of MilliQ dH20 was added to the
outer wells surounding the test wells in order to minimize evaporation.
Samples in a final
volume of 100 1/well were added to the 96-well plate as follows:
Table 2: Thioflavin T Fluorescence Assay
Sample No. Contents
1 P-Amyloid 1-40 peptide
2 P-Amyloid 1-40 peptide and Melatonin
3 P-Amyloid 1-40 peptide; Melatonin ; and ApoE4
4 P-Amyloid 1-40 peptide and Example 1
P-Amyloid 1-40 peptide; Example 1 and ApoE4
6 P-Amyloid 1-40 and Example 2,
7 P-Amyloid 1-40; Example 2; and ApoE4
[00243] Ratios
of components added to each well were as follows: 601LEM p-
Amyloid 1-40: 601LEM Melatonin or Example 1 or Example 2: 0.727[EM ApoE4 or
121LEM p-
Amyloid 1-40: 121LEM Melatonin or Example 1 or Example 2: 0.145[EM ApoE4.
Aggregation
was measured at 0 and 24 hours incubation time.
[00244] 5 [IL
was obtained from each test sample after incubation and added to
2 mL of a glycine/ NaOH buffer (50 mM, pH 9.2) containing 3 [tM thioflavin T.
Fluorescence intensities were measured at an excitation wavelength of 450 nm
and an
emission wavelength of 485 nm in a Envision fluorescence spectrophotometer. A
time scan
of fluorescence intensity was performed, and three measurements were taken
after the decay
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reached a plateau at 200, 220, and 240 s and averaged after subtracting the
background
fluorescence of 3 uM thioflayin T in the blank buffers. Neither melatonin nor
any of the other
compounds used in this study exhibited significant fluorescence within the
regions of interest
at any time point. All measurements were taken in triplicate. Siliconized
polypropylene
microcentrifuge tubes (USA Scientific) were used for these experiments.
Solutions of
recombinant apolipoproteins were immediately lyophilized and, prior to being
used,
resuspended in 0.1 M tris-phosphate-HC1 buffer (pH 7.4). Aqueous stock
solutions of 1 mM
melatonin were made by first preparing a 10 mM suspension of the hydrochloride
salt of the
hormone in 1 N HC1 and then by completely dissolving it in 100 mM phosphate-
buffered
saline at pH 7.4 (1:10, y:y) and readjusting the pH to 7.4 with 1 N NaOH.
Solutions of AP
were prepared by dissolving 2.2 mg of the peptide in 1 mL of 50 mM buffer at
pH 9.6.
Aliquots (50 uL) of this solution were lyophilized and stored at -80 C until
they were needed
for the experiments. Working stock solutions of the peptide (concentration of
500 uM) were
prepared in HPLC-grade water immediately prior to the experiments. The absence
of
aggregates and amyloid fibrils in these solutions was verified by
spectrofluorometry. In the
experimental samples, AP was further diluted 1:1 with phosphate-buffered
saline (pH 7.4,
100 mM) to which melatonin and/or apoE4 or equivalent volumes of buffer
solution were
added. The final concentration of AP in each sample was either 60 or 12 uM,
and the
melatonin:AB:apoE4 molar ratios were 100:100:0.83.
Results
[00245] As shown
in Figure 2, none of the three compounds tested significantly
reduced fibrillogenisis over a period of 24 hours when incubated at equimolar
concentrations
with AP alone. However, all the compounds reduced fibrillogenesis when ApoE4
was added
to the mixtures. The amount of ApoE4 compared to the test compounds was about
1/80.
Surprisingly, Example 1 and Example 2 were about 2 fold more potent than
melatonin in
reducing fibrillogenis in each case by 75%. These data suggest that melatonin-
antibody
conjugates made by the same methods will likely also exhibit this activity,
and accordingly
have therapeutic potential and may be advantageous compared to treatments that
do not
reverse the profibrilogenic properties of ApoE4.
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Example 4: Oxygen Radical Absorbance Capacity Assay (ORAC Antioxidant Test)
Materials:
[00246]
Sodium Fluorescein was purchased from Invitrogen. 2,2'-Azobis(2-
amidinopropane) dihydrochloride (AAPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-
carboxylic acid (Trolox0), gallic acid, Epigallocatechin gallate (EGCG),
epigallocatechin
(EGC), and quercetin dihydrate were purchased from Sigma-Aldrich (St. Louis,
Mo). Black-
sided, special optics clear bottom plates (part # 3615) were obtained from
Corning.
Background:
[00247] The
ORAC assay depends on the free radical damage to a fluorescent
probe, such as fluorescein, to result in a downward change of fluorescent
intensity. The
assumption is that the degree of change is indicative of the amount of radical
damage. The
presence of antioxidants results in an inhibition in the free radical damage
to the fluorescent
compound. This inhibition is observed as a preservation of the fluorescent
signal. Reactions
containing antioxidants and or blanks are run in parallel using equivalent
amounts of a
molecule capable of generating super reactive oxygen species (ROS) and
fluorescent probe.
Because the reaction is driven to completion, one can quantitate the
protection by calculating
the area under the curve (AUC) from the experimental sample. After subtracting
the AUC
from the blank, the resultant difference would be the protection conferred by
the antioxidant
compound. Comparison to a set of known standards allows one to calculate
equivalents and
compare results from different samples and experiments. Typically Trolox0, (6-
hydroxy-
2,5,7,8-tetrametmethylchroman-2-carboxylic acid) a water soluble vitamin E
analog, is used
as the calibration standard and ORAC results are expressed as Trolox0
equivalents. The
ORAC assay is unique in that because the assay is driven to completion the AUC
calculation
combines both the inhibition time as well inhibition percentage of free
radical damage by the
antioxidant into a single quantity.
Procedure:
[00248]
Reactive oxygen species generators were added to parallel reactions
containing equal amounts of a fluorescent probe. Reactions contained either a
buffer blank or
antioxidant samples and standards. The antioxidant capacity of a sample is the
net difference
between the area under the curve (AUC) of the sample and that of the blank.
The exterior
wells were not used for experimental determinations. 200 L of MilliQ dH20 was
added to
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the outer wells surrounding the test wells in order to minimize evaporation.
1504, sodium
fluorescein was added to experimental wells. In addition, blank wells received
25 11,1 of 75
mM phosphate buffer (BLANK), standards received 254, Trolox dilution (STD) and
samples received 254, Melatonin in 75mM phosphate buffer pH 7.4 (SAMPLES) in
the
Optiplate-96F solid black plates (Perkin Elmer). The plate was allowed to
equilibrate by
incubating for 30 min in the Victor2v Microplate Reader (Perkin Elmer,
Waltham, MA) at
37 C. Reactions were initiated by the addition 254, of AAPH solution (free
radical initiator)
for a final reaction volume of 200 4 The fluorescence was monitored
kinetically in
Victor2V with temperature control reader for at least 80min with data taken
every minute.
Fast orbital shaking (0.5mm) for 10 sec was performed prior to each reading.
The
temperature was set to 37 C at all time and the reagents were treated as
light sensitive.
Results:
[00249] The
kinetic curves of several different concentrations of Trolox
standard demonstrates varying amounts of protection of fluorescein against
oxidation that
results in the loss of fluorescence. The highest concentration tested (100
,M) provided
virtually full protection for approximately 20 minutes, before fluorescence
intensity began to
diminish, while the lowest concentration tested (6.25 ,M) provided only
slight protection
above the buffer only control.
[00250] As shown
in Tables 3-5, the melatonin conjugates prepared in
Examples 1-2 were shown to exhibit better antioxidant activity than Melatonin.
Table 3: ORAC assay measurements for Melatonin
Concentration Trolox Melatonin
AUC Net AUC AUC Net AUC
0 6.65 0.00 6.65 0.00
0.390625 11.95 5.30 14.74 8.09
0.78125 14.90 8.24 20.89 14.24
1.5625 20.00 13.34 28.74 22.09
3.125 28.96 22.31 42.59 35.94
6.25 44.91 38.25 66.10 59.45
12.5 77.61 70.96 88.65 82.00
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25 96.34 89.68 93.12 86.47
50 98.05 91.40 94.75 88.10
100 95.82 89.17 91.48 84.82
[00251] Calculated Trolox0 equivalents for Melatonin: 1.65. Trolox0
equivalents are
calculated as follows:
AUC=(R1/R1) + (R2/R1) + (R3/R1) + .............. +(Rri/R1) (Eq. 1)
Where R1 is the fluorescence reading at the initiation of the reaction and Rn
is the last
measurement.
Net AUC=AUC(sample) - AUC(blank) (Eq. 2)
A standard curve was obtained by plotting the Net AUC of different Trolox0
concentrations
against their concentration resulting in linear relationship. The resultant
standard curve was
then interpolated to determine of antioxidant capacity of the samples and
reported as Trolox0
equivalence. To determine Trolox0 equivalents of each sample range the ratio
of the slope
TE(range of concentrations) = m(sample)/m(Trolox0) (Eq. 3)
Table 4: ORAC assay measurements for Example 1 conjugate
Concentration Trolox Example 1 conjugate
AUC Net AUC AUC Net AUC
0 6.43 0.00 6.43 0.00
0.390625 13.53 7.10 14.99 8.56
0.78125 15.01 8.58 20.01 13.58
1.5625 19.69 13.26 29.51 23.08
3.125 29.49 23.06 46.55 40.12
6.25 44.55 38.12 74.68 68.25
12.5 74.88 68.45 93.56 87.13
25 100.28 93.85 93.03 86.60
50 97.48 91.05 88.39 81.96
100 99.05 92.62 82.12 75.69
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[00252] Calculated Trolox0 equivalents for Example 1 conjugate: 2.02.
Table 5: ORAC assay measurements for Example 2 conjugate
Concentration Trolox Example 2 conjugate
AUC Net AUC AUC Net AUC
0 6.23 0.00 6.23 0.00
0.390625 11.49 5.27 13.97 7.75
0.78125 14.62 8.39 20.49 14.27
1.5625 19.52 13.30 28.06 21.84
3.125 28.51 22.29 45.62 39.39
6.25 43.41 37.18 71.79 65.56
12.5 73.04 66.81 94.48 88.26
25 98.61 92.39 97.13 90.91
50 100.67 94.45 97.12 90.90
100 97.58 91.35 96.55 90.33
[00253] Calculated Trolox0 equivalents for Example 2 conjugate: 1.97.
Prophetic Example 1: Preparation of NO1-0X2 Antibodies
[00254] Melatonin (Sigma) is conjugated to a chimeric antibody
targeted to the
N-terminus of A13 peptide (N01) (Ab) using an SMCC linker. SMCC is dissolved
in
dimethylacetamide (DMA) and added to the antibody solution to make a final
SMCC/Ab
molar ratio of 10:1. The reaction is allowed to proceed for 3 hours at room
temperature with
mixing. The SMCC-modified antibody is subsequently purified on a GE Healthcare
HiTrap
desalting column (G-25) equilibrated in 35 mM sodium citrate with 150 mM NaC1
and 2 mM
EDTA, pH 6Ø The drug (e.g. melatonin) is dissolved in DMA, and added to the
SMCC-
antibody preparation to give a molar ratio of melatonin to antibody of 10:1.
The reaction is
allowed to proceed for 4 to 20 hours at room temperature with mixing. The
melatonin-
modified antibody solution is diafiltered with 20 volumes of phosphate-
buffered saline to
remove unreacted melatonin, sterile-filtered, and stored at 4 C. Typically, a
40% to 60%
yield of antibody is achieved through this process. The preparation is usually
greater than
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95% monomeric as may be assessed by gel filtration and laser light scattering.
Typically, the
drug to antibody ratio is expected to be between about 2.5 and 4.5. These same
conditions
can also be used to conjugate the constructs prepared in Examples 1-2 with a
chimeric
antibody targeted to the N-terminus of A13 peptide (N01) (Ab).
Prophetic Example 2: Confirmation of Reactivity of NO1-0X2 Antibodies
[00255] The ADC
prepared in Prophetic Example 1 is assayed to confirm
reactivity with the intended target epitope (N-terminus peptide of A13
peptide). The ADC are
subjected to Western blotting against each peptide of amyloid 13 (1-40),
amyloid 13 (2-40), and
amyloid 13 (3-40) according to known methods for carrying out Western
blotting. For
comparison, Chinese hamster cells in which amyloid 13 precursor proteins (APP)
are forcibly
expressed are homogenized with a buffer solution containing 1% Triton and
centrifuged to
obtain a supernatant liquid, which may be subjected to Western blotting in the
same manner
as above. This supernatant is expected to contain, in addition to APP, amyloid
13 and pc
terminal fragments (13CTF) cut from APP at the 13 site. In addition,
reactivity of each ADC
can be compared to that of unconjugated N01.
[00256] Binding
affinity of the ADC prepared in prophetic Example 1 may be
assayed using binding inhibition assays known in the art. See, e.g., Johnson-
Wood, K. et al.
(1997) Proc. Natl. Acad. Sci. USA 94:1550-1555.
Prophetic Example 3: In Vivo Assay for NO1-0X2
[00257] NO1-0X2
ADC obtained in Prophetic Example 1, unconjugated NO1
antibody, or melatonin alone is diluted with PBS to a concentration of 1 mg/ml
and
abdominally injected into 16-month old Tg2576 genetically engineered mice
exhibiting
amyloid precursor proteins (APP), at a dose of 10 mg/Kg (body weight) once per
week. As a
control, some mice receive only PBS administered in the same manner. After 12
injections,
mice are sacrificed and the left cerebral hemisphere is fixed with a 4%
formaldehyde buffer
solution and paraffin was embedded. 5 micron continuous segments are prepared
from the
paraffin-embedded cerebral tissue and immunologically stained with amyloid 13
(1-40)
polyclonal antibody and amyloid 13 (1-42) polyclonal antibody (both
manufactured by
Immuno-Biological Laboratories Co., Ltd., product numbers 18580 and 18582).
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[00258] Segments
from the cortical layer and from the hippocampus are
subjected to imaging analysis using a microscopic digital camera and simple
PCI software
(Compix, Inc. Imaging Systems, USA). Deposition of amyloid p (1-40) and
amyloid p (1-42)
is determined as the proportion (%) of the immunological staining positive
region in the
entire region.
[00259] In
addition, insoluble amyloid p in a 0.05 M Tris HC1 buffered
physiological saline solution (TBS, pH 7.6) is extracted from the frontal
right of the head (1/4
of the brain) with 6 M guanidine-hydrochloric acid to measure amyloid p (1-42)
using an
assay kit of amyloid p (1-42) and human amyloid p (1-42) (manufactured by
Immuno-
Biological Laboratories Co., Ltd., Product No. 17711) in a sandwich ELISA
system. The
method described by M. Morishima, Kawashima, et al., in Am. J. Pathol., 157
(2000) 2093-
2099 can be followed for extraction.
[00260] It is
expected that the number of senile plaques is smallest in the brain
of mice to which the N01-0X2 ADC was administered as compared to the other
treatment
groups. The number of senile plaques in the basal nuclei, in which the amyloid
p deposits is
slower than in the cortical layer or hippocampus, is also expected to be
smallest in N01-0X2
ADC treated mice. It is also expected that the amount of insoluble amyloid 0
extracted and
measured by ELISA, as described above, will be smallest in the IN-N01-0X2
treated group.
Prophetic Example 4: In vivo assay of NO1-0X2 in Blue Light AMD model
[00261] In this
Example, the ADC prepared in Prophetic Example 1 is tested
for its ability to protect retinal epithelial cells from oxidative damage.
This example evaluates
the prevention of photoreceptor damage following exposure to intense blue
light. This model
creates significant photo-oxidative stress and approximates the pathology
associated with
geographic atrophy, the late stage of Dry Age Related Macular Degeneration
(AMD).
[00262] 42
Sprague Dawley rats are acclimated to institution lighting for a
minimum of 21 days prior to light exposure. Light exposure is conducted over 2
sessions with
half of each study group challenged during each session (a total of 18 animals
are exposed to
blue light damage in each session). Prior to light exposure animals are dark
adapted
overnight. Ocular assessments begin after a minimum 5 day recovery period
following light
exposure. The details of this study are illustrated in Table 6 below.
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Table 6: Experimental Parameters of NO1-0X2 in Blue Light AMD model
Group N Test
2 10 Test Article 1:5 ul intravitreal injection both eyes on Day
-2
3 10 Test Article 1:5 ul intravitreal injection both eyes on Day
-2
4 8 Vehicle: 1:5 ul intravitreal injection both eyes on Day -2
8 8-0H-DPAT 5mg/kg. Once daily IP on Days-2 through Day +2
6 3-6 Untreated, unexposed Controls
[00263] Optical
Coherence Tomography (OCT) imaging is conducted on all
animals at Day >7 following a recovery period from light exposure. OCT and
fundus images
are collected using the Spectralis HRA+OCT (Heidelberg Engineering). Corneas
are
anesthetized by topical administration of 1-2 drops of Proparacaine and pupil
dilation is
achieved with 1-3 drops each of 1% Tropicamide and 2.5% Phenylephrine. The
animals are
anesthetized with isoflurane to effect. Four OCT slices are collected from
each eye,
approximately 150 from the Optic Nerve in the superior nasal, superior
temporal, inferior
nasal, and inferior temporal quadrants. Thickness measurements are made from
each
quadrant.
[00264]
Electroretinography (ERGs) is performed on all animals at Day >8
following OCT measurements. The ERG exam is conducted using the Espion E2 ERG
recording system (Diagnosys, LLC). Corneas are anesthetized by topical
administration of 1-
2 drops of Proparacaine and pupil dilation is achieved with 1-3 drops each of
1%
Tropicamide and 2.5% Phenylephrine. Dark adapted ERG's are recorded according
to
established protocols.
[00265] Any
chemical modification of the payload requires careful evaluation
to determine the effect of the modification. In addition, it is necessary to
evaluate the effect of
linking the compound to cysteine or lysine residues depending on which amino
acid is used
to conjugate the compound to antibody. Thirdly, it is necessary to evaluate
the
neuroprotective properties after conjugation to the antibody. Finally,
it is necessary to
demonstrate that the compound and method of conjugation do not significantly
affect the
affinity of the antibody or cause it to aggregate.
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[00266]
Depending on the mechanism of action of the payload, one would
evaluate its neuroprotective functions using one or more assays described in
Table 7.
Table 7: Evaluation of Payload
Properties Measured Assay
Free radical scavenging activity Free radical scavenging activity assays:
e.g. Oxygen radical absorbance capacity
assay
Reduced oxidative stress in vivo Evaluate markers of oxidative stress in
brain
of transgenic models (AD, HD & PD) by
ELISA assays and immunohistochemistry
a. iPF2-VI (a reliable biomarker of lipid
peroxidation)
b. protein carbonyls (known biomarkers
of protein oxidation)
Reduced inflammation in vivo Evaluate markers of inflammation in
brain of
transgenic models (AD, HD & PD) by ELISA
assays, Reverse transcription PCR and
immunohistochemistry
a. IL-113
b. IL-6
c. TNFa
d. YMI
e. AG1
f. GFAP
g. Mrcl and CD163 known to be
restricted to cerebral vasculature
Inhibition of fibrillogenesis Thioflavin assay
Reduced amyloid production Western blot analysis in supernatants
from
neurons in culture
Reduced amyloid deposits in vivo Quantitative immunohistochemistry in
fixed
tissue sections of transgenic mice or ELISA of
brain homogenate to measure effect on
amyloid deposition
Promotion of neurogenesis ELISA based format to measure effects on
neural differentiation potential quantified by
the use of three validated lineage specific
monoclonal antibodies, anti-bIII-tubulin for
neurons, anti-glial fibrillary acidic protein
(GFAP) for astrocytes and anti-CNPase for
early oligodendrocytes, in an ELISA based
format. Also included are control mouse IgG
and an anti-GAPDH monoclonal antibody to
correct for background signals along with a
cell stain solution to help normalize for
variations in the cell numbers across samples.
Cells are cultured and fixed in a standard 96
well plate with the lineage analysis done
directly on the whole cell population.
Blocked hyperphosphorylation of tau Immunocytochemistry or ELISA with
phospo tau antibodies
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Improved cognitive function Behavioral assays in transgenic models
(e.g.
Morris-Water Maze, Fear Conditioning, Y-
maze, Radial Arm Maze
Increased glucose utilization in the brain 1817-7-1MG 1PET metabolic imaging
of glucose
uptake or autoradiography of fixed tissue
sections from transgenic models
[00267] The
present invention is not to be limited in scope by the specific
embodiments described herein. Indeed, various modifications of the invention
in addition to
those described herein will become apparent to those skilled in the art from
the foregoing
description. Such modifications are intended to fall within the scope of the
appended claims.
[00268] All
patents, applications, publications, test methods, literature, and
other materials cited herein are hereby incorporated by reference in their
entirety as if
physically present in this specification.
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